Synthesis and Conformational Analysis of Jasplakinolide
Analogues and Approach towards the Synthesis of the
Stereotetrad of Cruentaren A
Synthese und Konformations Analyse von
Jasplakinolidanaloga sowie ein Vorschlag zur Synthese der
Stereotetrade von Cruentaren A
DISSERTATION
der Fakultät für Chemie und Pharmazie
der Eberhard-Karls-Universität Tübingen
zur Erlangung des Grades eines Doktors
der Naturwissenschaften
2006
vorgelegt von
Srinivasa Marimganti
Tag der mündlichen Prüfung:
23.11.2006
Dekan:
Prof. Dr. L. Wesemann
1. Berichterstatter:
Prof. Dr. M. E. Maier
2. Berichterstatter:
Prof. Dr. Th. Ziegler
This doctoral thesis was carried out from August 2003 to May 2006 at the Institut für
Organische Chemie, Fakultät für Chemie und Pharmazie, Eberhard-Karls-Universität
Tübingen, Germany, under the guidance of Professor Dr. Martin E. Maier.
First of all, I would like to thank Prof.Dr. Martin E. Maier for his excellent guidance during
my Ph.D period in Tüebingen. Whenever I want to discuss with him, with his limitless
patience he never denied me how much ever busy with his schedule. In such a wonderful
atmosphere I had enough time to achieve my goal without any problem. His teaching
assistance in every little aspect about synthetic organic chemistry, generous advice and
constant encouragement and limitless patience helped me to improve my knowledge in
various aspects.
I would like to thank Prof. Dr. Thomos Ziegler for his helpful reviewing the doctoral thesis
and for giving valuable comments and suggestions and Prof. Mariappan Periasamy for his
valuable suggestions during my Master’s Degree.
I personally thank Dr. Florenz Sasse for testing the biological activity, Mr. Graeme Nicholson
for his skillful technical assistance in various measurements, Mrs. Munari for preparing the
absolute solvents and for her kind help in the laboratory.
I thank all my working group members for their valuable discussions, suggestions and their
friendly nature. I specially thank Dr. Rajamalleswaramma Jogireddy, Dr. Andreas Petri,
Evgeny Prusov, Anton Khartulyari, Jan Ritschel, Markus Ugele, Dr. Manmohan Kapur and
Mrs. Naiser for their valuable suggestions in various aspects.
I would like to thank Murthy, Mahesh, Bhavani, Vikram, Surya, and Vamshi for their
tremendous support during my research period.
I would like to pay tribute to the constant support of my parents and brother for their love,
understanding and encouragement throughout my life. I don’t have words to explain about my
wife Prativa, I could say without her I might have not finished my Ph. D. in time.
for prativamayee
Publications:
S. Marimganti, S. Yasmeen, D. Fischer, M. E. Maier, Synthesis of Jasplakinolide Analogues
containing a Novel ω-Amino Acid. Chem. Eur. J. 2005, 11, 6687-6700.
S. Marimganti, M. E. Maier, Synthesis and Conformational analysis of Geodiamolide
Analogues Eur. J. Org. Chem. submitted.
Poster presentations:
S. Marimganti, M. E. Maier, Synthesis and Conformational analysis of Geodiamolide
Analogues containing Novel ω-amino and hydroxy acids. 9th International SFB-Symposium
SFB-380, Aachen, Germany, October 10-11, 2005.
Table of Contents
Table of Contents
Chapter I
1 Introduction ............................................................................................................................1
2 Literature Review ...................................................................................................................5
2.1 Summary of biological activity of Jasplakinolide and other related compounds......5
2.2 Modeling the jasplakinolide binding site on actin filaments .......................................7
2.3 The family of Jasplakinolide...........................................................................................8
2.4 Conformational (peptidomimetical) studies of small peptides ..................................11
2.4.1 Secondary Structure Motifs: β turns.........................................................................13
2.4.2 Sugar Amino acids (SAA)........................................................................................14
2.4.3 β-Amino acids ..........................................................................................................17
2.4.4 N-Methyl amino acids (NMA) .................................................................................18
2.4.5 Non bonded interactions...........................................................................................19
2.5 Key reactions and mechanisms ....................................................................................23
2.5.1 Stereo selective alkylation’s using chiral auxiliaries................................................23
2.5.2 Enzymatic hydrolysis using Pig liver esterase (PLE, EC 3.1.1.1)............................27
2.5.3 Formation of protected amines (carbamates) via Curtius Rearrangement ...............31
2.5.4 Ireland-Claisen Rearrangement ................................................................................35
2.5.5 Syn selective Evans Aldol reaction...........................................................................39
2.5.6 Esterification using the Yamaguchi method and DCC/DMAP conditions ..............43
2.5.7 Coupling reagents in solution phase peptide synthesis ............................................47
3 Goal of research ....................................................................................................................53
4 Results and Discussion .........................................................................................................55
4.1 Design of the novel ω-amino and hydroxy acids.........................................................55
4.2 Retrosynthetic analysis and synthetic pathways for amino and hydroxy acids ......58
4.2.1 Retrosynthetic analysis of amino acid 95 .................................................................58
4.2.2 Synthesis of amino acid 95.......................................................................................60
4.2.3 Synthesis of jasplakinolide analogues by incorporating the amino acid 95 .............65
4.2.4 Retrosynthetic analysis of hydoxy and amino acids 96 and 97................................68
4.2.5 Synthetic pathway for hydroxy acid 96....................................................................69
4.2.6 Retrosynthetic analysis of amino acid 97 .................................................................73
4.2.7 Synthetic pathway for amino acid 97 .......................................................................74
4.2.8 Synthesis of jasplakinolide (geodiamolide) analogues using 96 and 97 ..................77
4.2.9 Synthesis of Nor-methyl Chondramide C 185 .........................................................88
4.2.10 Design and the synthesis of analogues of hydroxy acid 96....................................93
4.2.11 Retrosynthetic analysis and synthesis of amino acid 194.......................................94
4.2.12 Synthesis of hydroxy acid 195 ...............................................................................98
4.3 Conformational studies ...............................................................................................100
4.3.1 Conformational studies of jasplakinolide analogues 129, 134, 135 and 136 .........100
4.3.2 Conformational studies of geodiamolide analogues 159 and 160..........................105
Table of Contents
Chapter II
5 Introduction ........................................................................................................................ 110
6 Synthesis of stereo tetrad 212 ............................................................................................ 112
6.1.1 Retrosynthetic analysis of stereo tetrad 212........................................................... 112
6.1.2 Synthesis of aldehyde 215...................................................................................... 113
6.1.3 Second retrosynthetic pathway for epoxide 212 .................................................... 115
6.1.4 Synthetic pathway .................................................................................................. 116
7 Summary and Conclusion.................................................................................................. 122
8 Experimental Section ......................................................................................................... 128
8.1 General Remarks......................................................................................................... 128
8.1.1 Chemicals and Working Techniques...................................................................... 128
8.1.2 NMR-spectroscopy................................................................................................. 128
8.1.3 Mass Spectrometry ................................................................................................. 129
8.1.4 Infrared Spectroscopy............................................................................................. 129
8.1.5 Polarimetry ............................................................................................................. 129
8.1.6 Melting Points ........................................................................................................ 130
8.1.7 Chromatographic Methods ..................................................................................... 130
8.1.8 Experimental procedures........................................................................................ 130
9 Appendix ............................................................................................................................. 204
9.1 NMR-Spectra for important compounds .................................................................. 204
9.2 Bibiliography ............................................................................................................... 244
Abbreviations
Abbreviations
abs.
absolute
Ac
Acetyl
arom.
aromatic
BBN (9-)
9-Borabicyclo[3.3.1]nonane
Bn
Benzyl
br
broad (NMR)
Boc
tert. Butoxy carbonyl
Bu
Butyl
BuLi
Butyllithium
c
Concentration
COSY
Correlation Spectroscopy
CSA
Camphor sulfonic acid
δ
Chemical shift in ppm (NMR)
d
Doublet (NMR)
DCC
Dicyclohexylcarbodiimide
DCM
Dichloromethane
de
Diastereomeric excess
DEPT
Distortionless Enhancement by Polarization Transfer
DIBAL
Diisobutylaluminium hydride
DMAP
4-Dimethylaminopyridin
DMF
N,N-Dimethylformamide
DMSO
Dimethylsulfoxide
dr
Diastereomeric ratio
E
trans
EDC
1-Ethyl-3-(3’-dimethylaminopropyl)carbodiimide
ee
Enantiomeric excess
EEDQ
2-Ethoxy-1-ethoxycarbonyl-1,2-dihydro-quinoline
EI
Electron impact
eq
equation
ESI
Electronspray ionization
Abbreviations
Et
Ethyl
Et2O
Diethyl ether
EtOAc
Ethyl acetate
Fig.
Figure
g
gram (s)
GC
Gas chromatography
h
hour(s)
HOBt
Hydroxybenzotriazole
HPLC
high performance liquid chromatography
HRMS
high resolution mass spectrometry
Hz
hertz
IR
Infrared
i-Pr
isopropyl
J
coupling constant
L
liter(s)
LA
Lewis acid
LAH
Lithium aluminium hydride
LDA
Lithium diisopropylamide
m
Multiplet (NMR)
Me
Methyl
MeOH
Methanol
mg
milligram
m/z
Mass to charge ratio (MS)
NMO
N-Methylmorpholin-N-Oxide
NMR
Nuclear magnetic resonance
NOE
Nuclear Overhauser Effect
NOESY
Nuclear Overhauser Effect Spectroscopy
PE
Petroleum ether
Ph
Phenyl
PLE
Pig liver esterase
PMB
p-Methoxybenzyl
PMP
p-Methoxyphenyl
Abbreviations
PPTS
Pyridinium para-toluene sulfonate
Py
Pyridine
PyBoP
Benzotriazole-1-yl-oxy-tris-pyrrolidino-phosphonium hexafluorophosphate
PyBroP
Bromo-tris-pyrrolidino-phosphonium hexafluorophosphate
q
Quartet (NMR)
Rf
Retention factor (TLC)
RT
Room temperature (ca. 23 °C)
s
Singlet (NMR)
s
Second
SAA
Sugar aminoacid
t
Triplet (NMR)
TBAF
Tetrabutylammonium fluoride
TBDMS
tert-Butyldimethylsilyl
TBTU
N-[(1H-benzotriazolo-1-yl)(dimethylamino)methylene]-Nmethylmethanaminiumtetrafluoroborate N-oxide
THF
Tetrahydrofuran
TfO
Trifluoromethanesulfonate
TPAP
Tetrapropylammonium perruthenate
Triflate
Trifluoromethanesulfonate
UV
Ultraviolet
Z
cis
Chapter I
Synthesis and Conformational Analysis of Jasplakinolide
Analogues
Introduction
1
1 Introduction
Cells of organisms bacteria, lichens, fungi, plants, animals produce a large variety of
organic compounds. Anciently many substances were obtained e.g. food stuff, building
materials, dyes, medicines, and other extracts from nature. Plants and animals have provided
substances used for their biological activity to heal or to kill and became the foundation of folk
medicine. Recent natural product discovery and development of avermectin (anthelminthic),
cyclosporin and FK-506 (immunosuppressive), mevinolin and compactin (cholesterol
lowering), and taxol and camptothecin (anticancer) have revolutionized therapeutic areas in
medicine.[1]
Vastly diversed living organisms of the marine environment are a potential source of
new drugs for treatment of antibiotic infections and other deadly diseases. Marine life
comprises over half a million species. Due to their unique living environment as compared
with the terrestrial organisms, marine organisms produce a wide variety of metabolic
substances which often have various unprecedented chemical structures. Enzymes, lipids, poly
heterosaccharides as well as secondary metabolites from marine sources can be defined as
bioactive marine natural products. In recent years, an increasing number of marine natural
products have been reported to exhibit various biological activities such as antimicrobial,
physiological and pharmacological ones. Some metabolites have also been noted by their
significant toxicities.[2, 3] The effort to find novel antitumor substances from organisms have
been increased in recent years and several novel compounds (peptides, polyethers, alkaloids,
prostanoids etc.), with antitumor activities, have been isolated from marine sponges,
octocorals, algae, tunicates, nudibranchs, bryozoans and so on. Several compounds from
marine invertebrates have reached clinical or pre-clinical anticancer trials. They include
bryostatin, didemnin B, dolastatin, ecteinascidin, halichondrin B, and elutherobin. Many other
compounds with different skeletal structures have also been bioassayed for antitumor activity.
Juncusol from an estuarine marsh plant, aplysistatin from a sea hare and aeroplysinin-1 from
sponges are only a few of the marine isolates which are undergoing further evaluation for
anticancer activity. Table 1 summarizes the reports on antitumor research, involving 14 marine
2
Introduction
compounds with determined mechanisms of action that included in vitro and/or in vivo studies
with human cancer cell lines.[4]
Table 1: Antitumor pharmocology of marine natural products with determined mode of
action
Compound
Organism
Chemistry
Agosterol
Sponge
Terpene
Aplidine
Tunicate
Depsipeptide
Auristastatin
Synthetic.
Peptide
Bryostatin
Bryozoa
Macrolide
Curacin D
Alga
Polyketide
Dehydrothyrsiferol
Alga
Terpene
Dolastin 10
Tunicate
Peptide
Ecteinascidin
Tunicate
Quinoline
Eleutherobin
Coral
Terpene
Jasplakinolide
Sponge
Peptide
Naamidine A
Sponge
Imidazole
Octalactin
Bacteria
Polyketide
Sarcodictyins
Coral
Terpene
Spirulan
Alga
Polysaccharide
Currently, about half of all described medicines are extracted or derived from terrestrial plants
and organisms. Many synthetic drugs were originally inspired by novel compounds in
terrestrial organisms. Although few marine natural products are currently in the market or in
clinical trails, marine organisms represent the greatest unexploited source of potential
pharmaceuticals. Because of the unusual diversity of chemical structures isolated from marine
organisms, there is an intense interest in screening marine natural products for their
biomedical potential. New drug discoveries indicate that marine organisms have a tremendous
potential for new pharmaceuticals and will become a much more prolific source than any other
Introduction
3
group of terrestrial organisms. The cyclodepsipeptide jasplakinolide (1) is a notable example
of the marine toxins that shows promising biological activities.
OH
O
Br
O
HN
HN
N
O
O
NH
O
Jasplakinolide (1)
Figure 1.1: Structure of marine natural product jasplakinolide 1
Jasplakinolide (Figure 1.1) was isolated from the marine sponges Jaspis splendens.[5]
Jasplakinolide was first described as having anthelminthic, antifungal properties.[6] It
represents a new class of bioactive cyclic depsipeptides. The structure of jasplakinolide
contains both peptide and polypropionate units as shown in Scheme 1. It is a part of a growing
family
of
structurally
related
other
natural
products
like
geodiamolides
A-F,
neosiphoniamolide, chondramides, doliculide, with the first two having the same
polypropionate structure as jasplakinokide, the 8-hydroxy acid 3 (Scheme 1). This hydroxy
acid contains four methyl groups in 1,3-distance giving rise to two syn-pentane interactions
and one 1,3-allylic interaction. Jasplakinolide and the other mentioned cyclodepsipeptides
possess very high activity against a number of tumor-derived cell lines, and therefore appeared
to be very promising candidates for cancer therapy. This has made them very attractive for
further biological investigation and structure activity relationship (SAR) studies.
4
Introduction
OPG
OH
O
Br
N
Br
O
HN
HN
O
HN
O
O
OR1
OR2
HN
O
O
N
NH2
NH
+
HO
O
O
1
2
3
Scheme 1: Tripeptide and polypropionate parts of jasplakinolide
Restricting the conformational flexibility of medium-sized polypeptides has proven a
valuable approach towards understanding the structural and conformational features of
bioactivity. Thus, in the case of peptide hormones, a number of studies in the past 20 years
have shown that this approach can lead to the discovery of analogues with potential agonistic
or antagonistic properties. The rationally designed conformationally constrained analogues are
in both cases tools for understanding the structure activity relationships of the natively
occurring polypeptides and lead structures for the development of novel compounds with
therapeutic and diagnostic applications.
In this direction, our target was focused on the design and synthesis of novel ω-aminoand hydroxy acids which have the similarity to the jasplakinolide polypropionate part 3 and to
synthesize novel analogues of jasplakinolide using these amino- and hydroxy acids.
Furthermore it was planned to study their solution conformations as well as biological
activities to gain some information concerning their structure-activity relationships. The ωamino- and hydroxy acids were designed on the basis of non bonded interactions such as 1,3allylic strain and syn-pentane interactions to get the restricted conformations for the analogues.
Literature Review
5
2 Literature Review
2.1 Summary of biological activity of Jasplakinolide and other related
compounds
Jasplakinolide (jaspamide), was first reported in 1986 as a novel biological active
cyclodepsipeptide.[5, 7] It was isolated from the Indo-Pacific marine sponge Jaspis johnstoni
(order Astrophorida), now known as Jaspis splendens or Jaspis sp. Quite unexpectedly, it was
reencountered during bioassay-guided isolations with extract fractions from Auletta cf.
constricta (order Halichondrida) possessing in vitro cytotoxicity to HT-29 cells. Jasplakinolide
was also shown to possess potent antiproliferative activity[8] in the NCI-60 cell line screen,
which prompted extensive further investigation of its properties and mechanism of action.
Additional studies demonstrated jasplakinolide to be especially active against a number of
tumor derived cell lines, human prostate carcinoma, and myeloid leukemia.[9] This drug was
observed to be active in vivo against Lewis lung carcinoma and human prostate carcinoma
xenografts.[10] A number of investigators have found jasplakinolide to be an invaluable tool to
probe cytoskeletal proteins and to observe the role of actin microfilaments. For example, Bubb
et al. found that jasplakinolide induces actin polymerization in a similar fashion to phalloidin.
The results imply that jasplakinolide exerts its cytotoxic effect by inducing actin
polymerization and/or inhibiting the depolymerization of muscle actin filaments.[11, 12] Actin is
an extremely conserved protein which forms the cytoskeleton in all eukaryotic cells. The
cytoskeleton is a dynamic three-dimensional structure that fills the cytoplasm. This structure
involved in movement and stability of the cell. The long fibers of the cytoskeleton are
polymers of subunits. The primary types of fibers comprising the cytoskeleton are
microfilaments, microtubules, and intermediate filaments (Figure 2.1.1).
Figure 2.1.1 a) Intermediate filaments
a
b
c
b) Microtubules
c) Microfilaments (actin)
6
Literature Review
Microtubules are cylindrical tubes, 20-25 nm in diameter. They are composed of
subunits of the protein tubulin, these subunits are termed alpha and beta. Microtubules act as a
scaffold to determine cell shape, and provide a set of "tracks" for cell organelles and vesicles
to move on. Microtubules also form the spindle fibers for separating chromosomes during
mitosis. When arranged in geometric patterns inside flagella and cilia, they are used for
locomotion. Actin is a globular structural protein that polymerizes in a helical fashion to form
an actin filament (or microfilament). Actin filaments provide mechanical support for the cell,
determine the cell shape, enable cell movements and participate in certain cell junctions, in
cytoplasmic streaming and in contraction of the cell during cytokinesis. In muscle cells they
play an essential role, along with myosin, in muscle contraction. Cellular actin exists in two
forms: as monomeric actin (G-actin) and as filamentous actin (F-actin). Linking monomers
(G-actin) to each other, forming long thread like structures, creates filamentous actin called Factin. F-actin has a polar structure with a fast growing barbed (+) end and slow growing
pointed (-) end. The barbed end favored for polymerization and monomers (G-actin)
preferentially are added to this end in their ATP-form.[13] After incorporation in F-actin, ATP
hydrolyses to ADP and as such the monomer (G-actin) becomes less stable at the pointed end,
leading in turn to depolymerization at the pointed end. This process is called tread milling
(Figure 2.1.2). Like phalloidin, jasplakinolide seems to be bind in a cleft between two
monomers of F-actin at the pointed end and thus prevents the depolymerization by disturbing
the dynamic nature (steady state) of F-actin. In fact, it was demonstrated that exposure of
living cells to jasplakinolide results in the formation of multinucleated cells and disruption of
actin in these cells.[14-16]
Figure 2.1.2: Formation of F-actin and process of tread milling
Literature Review
7
2.2 Modeling the jasplakinolide binding site on actin filaments
Figure 2.2: Molecular docking studies of jasplakinolide bound to F-actin A) jasplakinolide
(purple) binds between adjacent monomers and stabilizes lateral interactions in the filament B)
Closeup of the same view as in A showing key actin residues that interact with jasplakinolide
(purple). C) Expanded view of pocket showing the side groups of residues in host actin that
interact with jasplakinolide, differences in parasite actin shown in brackets. His (195) in gold.
Computer simulation studies of protein-protein interactions provided information about
molecular interactions that occur during the actin nucleation and polymerization and these are
highly relevant to binding of small molecules. Recent modeling studies[17] described the
binding site for jasplakinolide in muscle actin filament. Jasplakinolide binds in a cleft between
two adjacent monomers bridging lateral interactions between adjacent monomers in the
filament (Figure 2.2). Notably, this same site is targeted by the related cyclic peptide
phallodin, despite the fact that two structures are not highly similar. Binding of jasplakinolide
bridges between long pitch strands of two monomers of F-actin, stabilizes the filament from
depolymerization. Small molecule docking studies make very clear predictions about the
residues on actin that are important for binding to jasplakinolide. Closer examination of the
jasplakinolide molecule docked to the actin filament reveals that the closest residues are D187,
T201-T202, R206 on one monomer and L176-R177-L178-D179 on the other (muscle actin
numbering). All of these contacts are within ~4 Angstroms and likely are sufficiently close to
interaction with the side groups of jasplakinolide (Figure 2.2 B).
8
Literature Review
2.3 The family of jasplakinolide
Cyclodepsipeptides are characterized by the presence of at least one ester bond because
they contain a hydroxy acid. Furthermore they contain unusual amino acids, which may be
extended, N- methylated, hydroxylated, or halogenated. Occasionally they contain fragments
from other biosynthetic pathways, for example polyketides. The substituents on the polyketide
fragment might be used as conformational controlling elements. Some illustrative examples of
cyclodepsipeptides are jasplakinolide (1), geodiamolide (4), chondramide C (5), doliculide (6)
(Figure 2.3.1). While jasplakinolide (1) has a 19-membered ring system, the chondramides and
geodiamolide feature a smaller, 18-membered ring. Nevertheless, the similarity between
jasplakinolide and the chondramides is quite high since they share essentially the same
tripeptide fragment. Thus, there is a β-amino acid, an N-methyl-tryptophan, and an alanine in
both depsipeptides. In the chondramides the ω-hydroxy acid is one carbon shorter than in
jasplakinolide. On the other hand, the ω-hydroxy acid is the same in jasplakinolide and
geodiamolide. A somewhat related biological activity is reported for the geodiamolides which
supposedly cause microfilament disruption.[18] These observations highlight the role of the
tripeptide fragment as determinant of the mode of action. Even though doliculide (6) seems to
be similar to geodiamolide (4), in its biological activity it is comparable to jasplakinolide and
chondramide C.
Literature Review
9
OH
OH
O
H
Br
H
N
O
O
H
19-membered
H
N
Me
N
O
O
N
jasplakinolide (1)
N
Me
N
H
18-membered
N
N
chondramide C (5)
O
O
O
O
H
O
O
H
I
HO
Me
N
N
O
O
O
O
N
geodiamolide (4)
OH
O
I
HO
H
O
Me
N
O
18-membered
doliculide (6)
O
N
H
16-membered
Figure 2.3.1 Structures of jasplakinolide and related depsipeptides.
In a recent study[19] of doliculide (6) it was shown that it can potently enhance the
assembly of purified actin and inhibit the binding of FITC (fluorescein isothiocyanate)
labelled phallodin to actin polymer, like as the jasplakinolide, chondramide C, and phallodin.
Treatment of cells with doliculide caused them to arrest at cytokinesis and caused substantial
rearrangement of intracellular F-actin.[19] Similarly, Sasse et al.[20] found that chondramide and
phallodin differed little in their ability to displace a fluorescent phallodin derivative from actin
polymer. These observations suggest nearly identical affinity of the four compounds
(doliculide (6), jasplakinolide (1), chondramide (5), phallodin) for actin polymer and
encouraged to search for a common pharmacophore among these peptides. From the
modelling studies (Figure 2.3.2), it can be noticed that almost every atom of doliculide
overlaps with atoms in either jasplakinolide, phallodin or chondramide C. Of particular note is
that the benzyl group of doliculide corresponds to the indole group of other peptides and that
the iodine atom of doliculide overlaps reasonably well with the bromine atom of
10
Literature Review
jasplakinolide. In short, the phenyl ring and isopropyl group of doliculide occupy the same
region of space as the indole and the phenyl rings of jasplakinolide.
Figure 2.3.2: Comparison of computational volume overlap structures of doliculide with
jasplakinolide, phalloidin, and chondramide C. The overlapping structures of doliculide with
jasplakinolide (left), with phalloidin (center), and with chondramide C (right) are shown with
the molecules separated but in the same orientation in space. For each vertical pair of
molecules, matching atoms within their van der Waals radii are shown as spheres
superimposed on the stick figure representations of each molecule. Carbon atoms are shown in
green, oxygen in red, nitrogen in blue, sulfur in yellow, and halides in magenta. Hydrogen
atoms are not shown.
Literature Review
11
2.4 Conformational (peptidomimetical) studies of small peptides
For a number of biologically active peptides the incorporation of conformationally
constrained amino acids has given rise to analogues with improved biological activities.[21]
Presumably, a constrained analogue that retains biological activity is represented in solution
Figure 2.4.1: Relation of conformation vs biological effect.[22]
by a set of conformations that is smaller than that of the native unconstrained peptide, and that
set of constrained analogue conformations in solution contains a larger fraction of the receptor
bound conformation(s). Indeed, in certain cases multiple substitutions of constrained amino
12
Literature Review
acids have given potent analogues with sufficient rigidity to display discrete solution
conformations that are suggestive of bioactive conformation. Peptides bearing conformational
constraints have often displayed superior stability to enzymatic degradation as well.[23] In
solution peptides exist as a variety of conformations that are in dynamic equilibrium with each
other. If a conformational restriction is introduced to the bioactive conformation of the
peptide, forms A and B (Figure 2.4.1) can not arise. Thus the interaction with alternative
receptors and peptidases is suppressed or does not occur. In this fashion a desired biological
effect can be obtained.[22] Figure 2.4.2 explains the analogue (peptidomimetic) drug design
principles.
Receptor
Scan peptide libraries for
binding affinity
Biologically active peptides
alanine scan
Critical residues
reduce size
Potential peptide agonist
or antagonist
activity assays
Active peptide lead
compounds
Molecular modelling of
Receptor and/or ligand
Define active core
D-amino acid scan
non coded amino acid scan
Define local and global conformational parameters
Cyclization, turn mimetics,
isostere replacement
Generate active constrained analogues
(peptidomimetics)
Conformational analysis
Physical studies
Identification of receptor residues
responsible for peptide recognition
Hypothesis of receptor-bound
conformation
Figure 2.4.2: Analogue (peptidomimetic) drug design principles.
Several possibilities exist for the synthesis of conformationally restricted and/or metabolically
stable peptidomimetics at the amino acid level. The systematic exchange of individual amino
acids by α-C alkylated, α-N alkylated, and D-amino acids is well established. In addition, α,β-
Literature Review
13
unsaturated, cyclic and β-amino acids as well as amino acids with sterically demanding side
chains may also be employed. The Table in the down describes the effect on conformation
with the modification on the amino acid.[24]
β-alkylation
side chain modification
R
H2N
CO2H
N-alkylation
α-alkylation
i-1
R
i
i+1
ψ
O
R
ϖ
H
Cyclization,
N
OH
α,β−dehydrogenation
N
H2N
φ
H
O
R
O
χ
representative diagram for φψϖχ angles
Modification
1. Backbone N-alkylation
conformational effect
φ, ψ, χ are constrained, facilitates cis-trans
isomerism.
2. Backbone C-alkylation
φ, ψ are constrained to a helical or
extended linear structure.
3. D-amino acid/proline substitution
Favors formation of β-turn structures.
4. Peptide bond isosteres
ω can be fixed at 0 or 180o, or allowed
greater freedom of rotation.
5. Cyclic amino acids
ω can be biased to 0 or 180o, φ, ψ are
biased towards formation of β-turns or
γ-turns, χ can also be affected.
6. Dehydroamino acids
Fix χ at 0 or 180o.
7. β-alkylation
Constrain χ, may also affect backbone
conformation.
2.4.1 Secondary Structure Motifs: β turns
So far a crucial disadvantage of modifications (above Table) is, that the resulting
conformation can not be predicted. As a result biophysical investigations are necessary to
obtain conformational-activity relationships for each and every derivative prepared. For this
reason it is desirable to have methods available that induce a specific target conformation. A
secondary structure mimetic is a building block that forces a defined secondary structure after
14
Literature Review
incorporation into a peptide. An ideal mimic will have a rigid scaffold that orients the
sidechain residues in the same direction as the natural peptide, while confering better
solubility and/or resistance to enzymatic degradation. β-Turns are the most frequently
mimicked protein secondary structures. β-Turns are defined as a tetrapeptide sequence where
the distance between α-Ci and α-Ci+3 is less than or equal to 7 Å (Figure 2.4.3). The turn can
be stabilized by chelation of a cation, such as Ca2+ or intramolecular hydrogen bonds. In linear
peptides turn arises mainly due to the tendency to form intra molecular hydrogen bonds. The
β-turn is a structural motif common to many biologically active cyclic peptides[25] and has
been postulated in many cases for the biologically active form of linear peptides. For this
reason it is a frequently imitated secondary structure.[26]
O
O
N
H
HN
O
NH 7
HN
o
A O
Figure 2.4.3: β-turn.
There are certainly several ways of constructing conformationally locked and well-defined
structures of analogues of natural cyclic peptides using different strategies by introducing
different amino acids which are having some key features to control the conformations. The
following summary describes the utility of different kind of modifications of peptides which
are having key features in controlling the conformations to give specific secondary structures
such as β-turns.
2.4.2 Sugar Amino acids (SAA)
Since proteins tend to confer their biological activity through small regions of their
folded structures, in principle their functions could be reproduced in much smaller designed
molecules that retain these crucial surfaces. Introduction of sugar amino acids (SAA) in
peptides can adopt secondary turn or helical structures and thus may allow to mimic helices or
Literature Review
15
sheets. Kessler et al.[27] explored the conformational influence of several SAA (Figure 2.4.5)
into different model and biologically active peptides. After studying the NMR, CD and
considering molecular modeling calculations, they came to the conclusion that sugar amino
acids (SAA) can cause both global as well as local constraints.
HO
OH
OH
H2N
CO2H
HO
H
H
HO
OH
HO
O
O
HN
H
HO CO2H
NH
Siestatin B (9)
Hydantocidin (8)
Neuraminic acid (7)
NH
NHAc
HO
OH O
Figure 2.4.4: Naturally occurring sugar amino acids.
O
O
HO
H
O
H
NH O
Fmoc
10
HOHO
O
O
H
NH O
Fmoc
11
O
Figure 2.4.5: Kessler’s β-sugar amino acids.[28]
Somatostatin, a 14 amino acid peptide containing a 38-membered macrocycle is a hormone
formed in the hypothalamus. Structure-conformation-activity studies had suggested that a βturn composed of Phe-Trp-Lys-Thr is important for biological activity. Much of reminder of
the hormone apparently functions as scaffold and can be replaced with simpler structural units.
Many simpler analogues were designed[29] and they proved to be very active against tumor cell
growth and were able to induce apoptosis.
16
Literature Review
Fig 2.4.6: Model of the bioactive conformation of somatostatin hexapeptide analogues: [cyclo(Pro-Phe-D-Trp-Lys-Thr-Phe)].
Kessler et al. replaced proline in the bioactive conformation (Figure 2.4.6) of the somatostatin
hexapeptide analogue with various sugar amino acids (SAA).[30] NMR and other studies
showed that there was a β-turn due to the SAA. Somatostatin analogues 12 and 13 containing
SAA 10 exhibit strong antiproliferative and apoptic activity against the multidrug resistant
hepatomic carcinoma. Results reveal that by introducing the SAA in a peptide backbone,
pharmacokinetic properties can easily be improved as well as bioavailability, and enzymatic
stability of the compounds will most likely also be enhanced.
O
O
O
O
O
O
O
NH
O
OH
HN
O
NH
O
H
N
HN
O
O
NH
OH
HN
O
NH
O
H
N
O
HN
O
O
O
HN
HN
H2N
H2N
13
12
Figure 2.4.7: Sugar amino acid 10 containing compounds 12 and 13 with antiproliferative and
apoptotic activity in the low μM range.
Literature Review
17
2.4.3 β-Amino acids
A peptidomimetic approach that has emerged in recent years with significant potential
is the use of β-amino acids. β-Amino acids are similar to α-amino acids in that they contain an
amino terminus and carboxyl terminus. β-Amino acids, with a specific side chain, can exist as
the R or S isomers at either α (C2) carbon or β (C3) carbon. This results, in a total of four
possible isomers for any given side chain. The flexibility to generate a vast range of stereo and
regio isomers, together with the possibility of disubstitutions, significantly expands the
structural diversity of β-amino acids thereby providing enormous scope for molecular design.
H2N
CO2H
H2N
CO2H
H2N
CO2H
H2N
CO2H
Figure 2.4.8: Structure of β-amino acids and the side chain stereochemistry of the four
possible isomers of a mono-substituted β-amino acid.
The incorporation of β-amino acids has been successful in creating peptidomimetics that not
only have potent biological activity, but are also resistant to proteolysis. In a recent study of
designing enzyme inhibitors of the endo-peptidase, scissile α-amino acid was replaced with a
β-amino acid against proteolysis. A likely mechanism for the stabilization of a peptide bond by
a β-amino acid involves the displacement of scissile bond from the active site due to the
presence of an additional carbon atom in the back bone and preventing proteolysis (Figure
2.4.9).[31]
18
Literature Review
Figure 2.4.9: Schematic diagram of the binding of an α-peptide (upper panel) and a β-amino
acid containing peptide (lower panel) to the active site of EP24.15 demonstrating how
a β-peptide may bind but is not cleaved by the peptidase.[31]
There are several biologically known active cyclic peptides containing β-amino acids.
Jasplakinolide, the chondramides contain the β-phenyl alanine and taxol contains a α-hydroxy
β-amino acid derivative. There are plenty of examples where an α-amino acid replaced by βamino acid resulting in higher biological activity and increased stability to proteolysis.[32, 33]
2.4.4 N-Methyl amino acids (NMA)
NMA containing peptide natural products (peptides, depsipeptides) have been isolated
from a variety of sources and their secondary metabolites (e.g. vancomycin, cyclosporin,
actinomycin D) have found clinical use due in part to physical properties and chemical
stability conferred by NMAs present in their structures.[34] Short poly N-methylated peptides
or peptides containing altering N-methyl amide and normal amide bonds have been especially
successful as inhibitors of amyloidosis ie the process of protein aggregation thought to be, at
least partially responsible for Alzheimer’s disease, type II diabetes etc.[35,
36]
(Amyloidosis
refers to the extracellular deposition of a protein called amyloid. This protein deposition can
affect multiple organs. The deposition of amyloid may be a byproduct of normal aging, or may
occur with several other conditions). Studies of NMA containing peptides reveal that N-methyl
amino acid residues increase the proteolytic stability, increase membrane permeability
(lipophilicity), and alter the conformational characteristics or properties of amide bonds.[37]
Literature Review
19
In 1967, Goodmann et al. proposed that poly N-methyl alanine adopts a helical
conformation by using low resolutions methods like circular dichroism (CD) spectroscopy,
one dimensional 1H NMR spectroscopy and some theoretical calculations. In contrast to this
work more recent literature, especially in biological journals, assumes that peptides containing
N-methyl amino acids prefer an extended conformation. The rationale for this is a report by
Viteroux et al. in which they studied the effect of N-methylation on the conformation of amide
bonds through the use of homo- and hetero-chiral dipeptides, in which one amide bond was Nmethylated. In this paper it was suggested that homo-chiral dipeptides with an internal Nmethylated bond prefer a cis-amide form, giving the peptide β-turn characteristics while
hetero-chiral dipeptides exist in the trans-amide form. Comparison of these effects with
dipeptides containing a proline residue concluded that the effect of N-methylation giving a
tertiary amide was less than the geometrical constraints conferred by proline residues.[38] More
recently Arvidsson et al.[39] studied the synthesis and crystal structures of poly N-methyl
alanine (penta and hexa) and hetero poly N-methyl peptide containing various α-amino acids.
In this paper they found that both homo poly N-methyl alanine and hetero poly N-methyl
peptide adopt an extended conformation (β-strand) and all amide bonds populated the trans
amide form which allows the formation of hydrogen bond between natural α-peptides. The
dihedral angle values revealed that, only every second residue can interact with the sheet
conformation of α-peptide. These results exposed that the peptides with alternating N-methyl
groups can inhibit the growth of β-sheet conformation of the natural α-peptide and thus can
inhibit amyloidosis.
2.4.5 Non bonded interactions
Most of the polyketides isolated from nature have very complex structures containing
methyl groups in different configurations. One can wonder why nature makes such
complicated molecules which are having many methyl groups in a symmetric or complex way.
Such considerations strengthen the notion that the presence of the numerous methyl side
groups is somehow connected with the backbone conformation of these molecules. The methyl
groups do not affect the flexibility of the backbone, yet they reduce the number of low energy
local conformers with the result that such molecules preferentially populate certain
20
Literature Review
conformations. In short, these polyketides may be called as ‘flexible molecules with defined
shape’.[40]
Two principles become evident, that nature uses to destabilize undesired conformations: one is
to avoid the 1,3-allylic interaction and the other is to avoid syn-pentane interactions. In a
substance as depicted in Figure 2.4.10, a single conformation of the vinylic bond is almost
completely populated in which the H-C-C=C dihedral angle lies within 0±30o. The eclipsed
interaction is consequently more costly (16.3 kJ mol-1) and the bond rotates in a way to release
the repulsive interaction as shown in Figure 2.4.10.
CH3
H3C
CH3 H
a
H
H
H
CH3
H3C
H
H3C
b
H
H
1,3 allylic interaction
Figure 2.4.10 Preferred local conformation of a 1,1-disubstituted allylic system
A destabilizing syn-pentane interaction is created when a hydrocarbon chain is folded such a
way that a gauche+ (60o) dihedral angle is followed by gauche- (300o) along the backbone as
illustrated in Figure 2.4.11. This places in the case of Figure 2.4.11, two methyl groups into a
similar spatial proximity, as formed in a 1,3-diaxial arrangement on a cyclohexane ring. The
conformation shown in Figure 2.4.11 is no minimum on the energy hyper surface; rather the
molecule relaxes by increasing the backbone dihedral angle. The resulting conformers are still
higher in energy (about 14 kJ mol-1) than unstrained conformers. For this reason, linear
hydrocarbon chains in alkanes adopt conformations that are free of such syn-pentane
interactions. The methyl side groups in the polyketide natural products cause the substructures
of the kind in Figure 2.4.11 to have only two conformations that are free from such
destabilizing interactions.[41, 42]
Literature Review
CH3
g CH3
-
21
R 2
6 R
H3C
g+
a
R
R
H3C
R
H
R
H
CH3
b
CH3
H
R
CH3
R
H
R
CH3
H
H
R
c
H
H3C
CH3
R
R
H
Figure 2.4.11 a) Destabilized by syn-pentane interaction; b, c) diconformational segments of
2,4,6… n-polymethylated alkane chains.
The 8-hydroxy acid (3) of jasplakinolide (1) is a good example, which fits in the definition
‘flexible molecule with defined shape’. The hydroxy acid 3 contains four methyl groups in a
1,3-distance. One can identify two syn-pentane interactions and one 1,3-allylic interactions.
Due to these interactions (because of methyl groups) both functional groups at both ends of the
chain point in one direction and allow bridging with a peptide fragment. Based on the work of
Hoffman et al. one can assume that the conformation of the chain is largely determined by the
central double bond and the three other methyl groups. While definitely several low energy
conformations are possible, an arrangement such as one depicted in Figure 2.4.12 should be
accessible. Conformational search runs (Macromodel 7.0, MM2* force field, 1000 starting
structures) for the hydroxy acid 3 found four conformers within 4.184 kJ mol-1 of the
minimum in which the third lowest conformer (ΔE = 2.00 kJ mol-1) matches the expected
conformation for hydroxy acid 3 depicted in Figure 2.4.12. This would allow an easy bridging.
The conformation of the central part is governed by 1,3-allylic strain. The dihedral angles of
the single bonds next to the allylic system are gauche+ (60o) and gauche- (300o),
respectively.[43]
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Literature Review
1,3-allylic interaction
1
CO2H
OH H
H3C
CH3
H
8
2
4
2,3 g+ 3,4 g+ 6,7 g- 7,8 gCH3
6
H
H3C
syn-pentane interaction
HO
CO2H
3
eclipsed
Figure 2.4.12: Possible conformation of the 8-hydroxy acid 3 due to the avoidance of CH3CH3 steric interactions.
In recent sequel publications, Terracciano et al.,[44,
45]
described the synthesis and
conformational analysis of simplified analogues of jasplakinolide (Figure 2.4.13). In the first
publication,[44] the polyketide part was modified by replacing the 8-hydroxy acid 3 with
commercially available 5-aminopentanoic acid, 6-aminohexanoic acid, 8-aminooctanoic acid.
In the peptide part D-N-methyl-2-bromotryptophane and D-β-tyrosine were replaced with Dtryptophane and L-valine basing on the theory discussed earlier in section 2.3, in order to gain
information about the pharmocophoric part of jasplakinolide. But there is no rational
conformational constraint within the analogues 14, 15 and 16. Moreover, there was no
significant biological activity. This may be due to the lack of conformational elements in the
polyketide part of these analogues. In the second publication,[45] hydroxy acid 3 was replaced
with substrate 17. Hydroxy acid 17 was designed by substituting the allylic part with a meta
substituted aromatic part in the place of allylic part and replacing one of the methylene group
in the chain with its isosteric oxygen. In addition, the methyl groups were removed in the
design process. Therefore, while showing some constraint, hydroxy acid 17 is lacking the
crucial syn-pentane interactions. The results revealed that there was neither good correlation in
the conformations of the analogues with the natural product and nor specific biological activity
towards the F-actin. There was no specific β-turn type II secondary structure for the analogues
in which the hydroxy acid was specifically designed to get secondary structure motifs. It
seems that in this publication, there was given more importance to the tripeptide part than the
polyketide fragment as it was thought that the tripeptide fragment is the key determinant for
exerting the biological activity.
Literature Review
23
H
N
HN
HN
HN
O
O
H
N
HN
O
HN
HN
O
O
O
N
H
N
H
14
15
OH
deleting the
methyl groups
HN
O
HN
HN
O
HO2C
O
O
O
O
NH
NH
O 16
HO
OH
ring closure by
aromatic ring
HO2C
H
N
replacing with isostere
3
O
HO2C
17
Figure 2.4.13: Jasplakinolide analogues (above); replacement of the allylic part with an
aromatic ring in the polyketide part of jasplakinolide.[45]
2.5 Key reactions and mechanisms
2.5.1 Stereo selective alkylation’s using chiral auxiliaries
A classical strategy to obtain enantiomerically pure compounds is based on the use of
chiral auxiliaries. Thus, a chiral, enantiomerically pure compound is attched to an achiral
starting material. This way enantiotopic groups or faces become diastereotopic. After the
diastereoselective reaction on the conjugate, removal of the auxiliary provides only the derived
enantiomer of the product. The versatile oxazolidine-2-one based chiral auxiliary was first
developed by Evans et al. in 1981.[46] The auxiliaries are easy to prepare from α-amino acids.
Reduction of the amino acid to the amino alcohol and subsequent cyclization using diethyl
24
Literature Review
carbonate gives the chiral auxiliary. For instance chiral oxazolodin-2-ones 19-21 and 23 are
available from valine, phenyl alanine, phenyl glycine, and norephedrine, respectively.[47, 48]
O
HO
NH2
O
R
1.LiAlH4 or BH3SMe2
NH
O
2. (EtO)2CO/K2CO3
19 R = CHMe2
= Bn
20
= Ph
21
R
18
O
HO
NH2
H3C
(EtO)2CO/K2CO3
NH
O
R
Ph
H3C
22
23
Scheme 3: Synthesis of chiral auxiliaries from the corresponding α-amino acids or amino
alcohols.
Evans chiral auxiliaries have been utilized in a wide variety of synthetic transformations[49]
such as asymmetric syn-aldol reactions, stereoselective alkylations, stereoselective
differentiation of enantiotopic groups in molecules bearing prochiral centers, asymmetric
Diels-Alder reactions, and the synthesis of β-amino acids etc.[50] Scheme 4 describes some
important reactions of propionyloxazolidinone 24 with various electrophiles.
Literature Review
25
O
LDA
Li
allyl bromide
N
O
O
O
O
O
N
96% de
H
Bn
O
O
O
N
O
H
O
N
Ph
NaHDMS
O
24
Na
O
Bn
O
SO2Ph
N
O
N
O
>90% de
Bn
H OH
Bn
R
O
KHDMS
O
K
O
SO2N3
O
N
Bn
R
R
O
O
N
R = iPr
Bn
>90% de
N3
Bn
Scheme 4: Different reactions using different bases and electrophiles on propionyl
oxazolidinone 24.
The high selectivity of alkylations using propionyl derivative 24 can be explained on the basis
of a selective enolate formation and approach of the electrophile from the less hindered side of
the enolate. Amides invariably give only Z-enolate upon treatment with bulky bases like LDA
(Figure 2.5.1) because the A(1,2) torsional strain dominates over the 1,3-diaxial interaction in
the chair like transition states of the deprotonation step.
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Literature Review
1,3-diaxial strain
R
R
Me
H
R
N
O
Li
N
R
O
Li
H
H
Me
little
A (1,2) strain
A (1,2) strain
(torsional)
LiO
N
H
N
LiO
N
N
Me
Me
Z-enolate
E-enolate
Figure 2.5.1: Possible explanation for the formation of Z-enolate.
There are two conformations possible for the propionyl derivative 24 and for the enolate 25.
But because of the chelation of oxygens with lithium, one conformation (Figure 2.5.2) is
highly favored due to the chelation. This way efficient differentiation of the two enolate faces
is guaranteed. Now the enolate attacks the electrophile from the less hindered side (Figure
2.5.3) forming the product with excellent diastereoselectivity for the compound 26.[50]
O
O
O
O
O
N
24 Bn
O
O
25
Li
Bn
O
O
N
Bn
O
N
O
N
O
Li
Bn
Figure 2.5.2: Possible conformations of chiral auxiliary and enolate.
Literature Review
27
R
Me
O
I
R
O
Li
O
Me
H
H
O
O
O
O
H
N
R
Bn
26
Ph
Ph
benzyl group blocks
enolate from the bottom
Figure 2.5.3: Attack of enolate from less hindered side.
2.5.2 Enzymatic hydrolysis using Pig liver esterase (PLE, EC 3.1.1.1)
Enzymes are now accepted as useful catalysts for a broad range of organic
transformations due to their capacities for inducing efficient asymmetric transformations.
Hydrolases are currently the enzyme group that receives the most attention. Among the
hydrolases, the esterase that has seen most extensive utilization is pig liver esterase (PLE, EC
3.1.1.1). PLE is a serine protease that catalyzes the hydrolysis of a broad range of carboxylic
acid esters. The potential of PLE in organic synthesis first emerged when Sih and coworkers
reported the PLE catalyzed enantioselective hydrolysis of diester 27 (Scheme 5). PLE is
capable of enantiomeric and enantiotopic group specificity and widely employed for providing
chiral acid ester synthons from prochiral diester substrates. In addition PLE can also be used to
resolve racemic mono esters.[51]
HO CH3
MeO2C
CO2Me
PLE, pH 8, 25 oC
3h
HO CH3
CO2H
MeO2C
27
28
HO CH3
HOH2C
HO CH3
CO2H
29
O
30
O
Scheme 5: Asymmetric hydrolysis of symmetric diester.
LiBH4
Na/NH3
28
Literature Review
Complications arose when PLE showed uncertainty about the stereochemistry towards certain
substrate groups. For example within the homologous series of monocyclic meso diesters 3133, the stereoselectivity of PLE hydrolysis reverses itself. For the cyclobutane diester 31, the
S-ester is hydrolyzed to give 34, while for the cyclohexane substrate 33, the acid-ester 36 from
R-ester cleavage is formed. Both 34 and 36 are enantiomerically pure. The cyclopentane
substrate 32 represents the changeover point, with the acid-ester 35 being virtually racemic.
Initially it was thought that the variability in stereoselectivity is due to the commercially
available PLE that contain the similar proteins with some having R- and other S- preference
(isoenzymes). The separated PLE components did not change the stereospecificity of 31-33,
thereby demonstrating that, although commercial PLE is a mixture, it behaves as if it is single
species.
CO2CH3
CO2CH3
PLE
31 CO2CH3
CO2CH3
34 CO2H
PLE
CO2CH3
()
CO2H
CO2CH3
32
35
CO2CH3
CO2H
PLE
CO2CH3
33
CO2CH3
36
2.5.2.1 Modeling the active site of PLE
Due to the uncertainty in the stereochemistry of PLE towards certain substrates and the
lack of an X-ray structure, an empirical approach was put forward, the active site model
(Figure 2.5.4) for PLE, as proposed by Toone et al.[51] in 1990. This model is a theoretical
approach using computer modeling studies and based on the analysis of experimental results
of PLE catalyzed hydrolysis of literature known substrates. First, the active-site location of the
catalytically vital serine residue involved in the ester hydrolysis was arbitrarily fixed in a
unique location in the space. Then using computer graphics, for each substrate the ester group
that becomes hydrolyzed was placed in the nucleophile region and the remaining parts of the
Literature Review
29
substrates were overlaid in order to identify the components that could occupy common
volumes. The analysis resulted in an illustrative active-site model (Figure 2.5.4) for PLE.
serine
PB
PB
HL
HS
PF
serine
HS
HL
Top-perspective view
A
PF
B
Figure 2.5.4 Active-site model for PLE.
The catalytically essential region is that of the serine residue, which initiates hydrolysis by its
attack on the carbonyl group of the susceptible ester function. The binding regions controlling
the specificity are composed of four pockets of which two are hydrophobic (HL(large) and
HS(small)) which interact with the aliphatic or aromatic hydrocarbon portions of a substrate and
two others that are more polar in character (PF(front) and PB(back)). The larger of two hydrophobic
zones, HL, has a volume of approximately 33 Å3, while the smaller HS pocket has a roughly
5.5 Å3. Polar groups such as hydroxyl, amino, carbonyl, nitro, etc. are excluded from these
areas. However, the hydrophobic pockets can accommodate less polar hetero atom functions
such as halogen, and ether, or ketal oxygen atoms, if necessary. The remaining two groups
accept more polar or hydrophilic groups. They are located at the front (PF) and back (PB) of the
active site, respectively. Unlike the other binding regions, the rear boundary is open, and
hydrogen bonding or similar groups may extend out beyond the back of this region. The area
above the model is also open, and is completely accessible to any substrate moiety that needs
to locate there. Such groups may extend in this direction without restriction. This model
reveals the differences in the stereochemistry of the homologous series 31-33 towards the
hydrolysis with PLE. Small hydrophobic groups bind in HS until they become too large to do
so, at which point the substrate orientation must turn around to place the large hydrophobic
groups in HL pocket, where there is room to accommodate it. It is this ‘turning over’
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Literature Review
requirement that is responsible for the S-to-R (and vice versa) switches observed
31
34
serine
CO
(a)
2 Me
experimentally. Figure 2.5.5 explains the reversal of stereochemistry for the series 31-33.
R
2C
S
32
MeO
CO
2 Me
serine
35
e
CO 2M
(c)
CO
S
2C
serine
e
M
36
2
R
CO
Me
O
33
serine
e
M
(d)
S
R
(e)
2
CO
S
R
favored
serine
e
M
(b)
2 Me
(f)
2
CO
serine
MeO2C
S
R
2M
e
favored
Me
O
S CO
2C
R
Figure 2.5.5: A top perspective view of the active-site model for the hydrolysis of the
homologous diester series 31-33.[52]
The top perspective view of the active site model is used to illustrate the binding-mode
selections for the diesters 31-33. Dimethyl cyclobutane-1,2-dicaroboxylate 31 bound into the
active site with its ‘S’ centre ester in the serine sphere and thus being hydrolyzed (Figure
2.5.5a). In this orientation the ‘R’ centre can locate acceptably within the PF pocket and the
cyclobutyl group is directed in to the HS site, where it fits well. This is clearly a favorable ‘ES’
complex. The alternative binding mode required for hydrolysis of the R-centre ester would
Literature Review
31
place a portion of the cyclobutane ring in the HL pocket. Since binding of hydrophobic groups
must occur in the HS rather than the HL site if sterically possible. It seems to be the
hydrophobic part that is cyclobutane ring sterically fits well in HS leading to the formation of
S-acid product 34. In the case of dimethyl cyclohexane-1,2-dicarboxylate 33 the binding
depicted in Figure 2.5.5e shows the preferred ES-complex for hydrolysis of the R-center ester
2R-acid 36, with the cyclohexane ring bound in the large hydrophobic pocket HL. Hydrolysis
of the ‘S’ centre would require an orientation as shown in Figure 2.5.5f. This is precluded
since the HS pocket is clearly too small to accept the six-membered ring. According to the
active-site model (Figure 2.5.5c and d), hydrolysis of dimethyl cyclopentane-1,2-dicarboxylate
32 is the intermediate between the two cases 31 and 33 since it can fit into both HS and HL but
the cyclopentane group is marginally too large for an optimum fit into HS, resulting in a slight
preference for hydrolysis on the R side (experimental result showed 17% ee) which satisfies
the experimental result.
2.5.3 Formation of protected amines (carbamates) via Curtius Rearrangement
The Curtius rearrangement (eq.1) involves the thermal decomposition of acyl azides
into amines via an isocyanate intermediate. The reaction might be explained with the loss of
nitrogen from the acyl azide forming an acyl nitrene species. Migration of the ‘R’ group to the
electron deficient nitrene would then form the isocyanate. The isocyanate can be trapped by a
variety of nucleophiles such as H2O, amines, or alcohols to get amines, acylureas, or
carbamates, respectively (eq. 1). Curtius rearrangement is one of the most widely used
methods to synthesize amine derivatives and over 1000 references can be found in the
literature related to the rearrangement which points to the potential of the reaction.[53] This is a
very general reaction and can be applied to any carboxylic acid: aliphatic, aromatic, acyclic,
heterocyclic, unsaturated, and other polyfunctionalized carboxylic acids.
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Literature Review
O
O
+
R
Cl
NaN3
O
-N2
R
N3
R
N C O
eq. 1
R
N
H2O
R'NH2
R'OH
R
H
N
H
N
R NH2
R'
O
R
H
N
O
R'
O
A number of methods have been developed to obtain acyl azides from carboxylic acid
derivatives, such as acyl chlorides, mixed anhydrides, and hydrazides. Normally, obtaining the
acyl chloride from carboxylic acid sometimes might cause racemization (if the compound has
a chiral centre next to carbonyl group) or decomposition. In 1961, Weinstock reported the
formation of acyl azides from the mixed anhydride which in turn can be obtained by treatment
of carboxylic acid with ethyl chloroformate (eq. 2).[54]
O
R
O
C
ClCO2Et / NEt3
OH
R
N
O
H
R
R
H
37
O
N
H
C
O
38
O
O
O
Et
O proton transfer
H
O
NaN3
R
H
N
C
O
O
eq.2
N3
R
CO2
H
R NH2
40
OH
39
carbamic acid
Scheme 6: Formation of amines from isocyanate.
Isocyanate 37 upon hydrolysis with H2O forms the amine via unstable carbamic acid
intermediate 39 as shown in Scheme 6. The carbamic acid 39 decomposes in presence of base
and gives amine 40.
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33
O
C
R
N
R
O
R'
37
H
N
R'
C
O
O proton transfer
R
H
N
H
42
C
O
O
R'
43
carbamate
Scheme 7: Formation of carbamate from isocyanate.
Isocyanate 37 upon treatment with alcohols produces the protected amines which are called
carbamates (Scheme 7). The mechanism is similar to that of Scheme 6, but here the
carbamates are stable as opposed to the carbamic acid. A variety of alcohols can be used to
produce the corresponding carbamates. For example t-BuOH produces the Boc-protected
amine, fluorenyl alcohol gives the Fmoc-protected amine and benzyl alcohol produces the
Cbz-protected amine.
In 1974, Yamada et al.[55,
56]
reported a modification of the Curtius rearrangement using
diphenyl phosphoryl azide (DPPA) (Scheme 8). Using this reagent carboxylic acids yield
amine derivatives (especially carbamates) in a single operation (does not require the acyl
chlorides or mixed anhydrides). Diphenyl phosphorizidate serves as azide source as well as the
coupling reagent, producing the mixed carboxylic phosphoric anhydride in the presence of
triethyl amine.
O
R
DPPA / NEt3
OH
44
R'OH, heat
R
H
N
C
O
45
O
R'
O
P
PhO
OPh
N3
DPPA 46
Scheme 8: Modified Curtius rearrangement.[57, 58]
The initial stage of the reaction involves the interaction of the carboxylate anion 47 with the
phosphorous atom of DPPA 46 by releasing the azide anion to form the activated carboxylic
phosphorus ester 48 (a mixed anhydride of carboxylic acid and phosphoric acid diester) as
shown in Scheme 9. Now the azide attacks back on the carbonyl carbon of 48 to form the
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Literature Review
carboxylic azide which on heating rearranges to the isocyanate according to same mechanism
as that of ordinary Curtius rearrangement (eq 1). DPPA became the most widely used reagent
for the one-pot synthesis of urethanes from carboxylic acids such as aliphatic, aromatic,
heterocyclic ones etc. because the reaction needs neither a strong acid nor a strong base and it
produces the acyl azide directly from the carboxylic acid without requirement of acid chlorides
or mixed anhydrides.
R
O
O
O
O
H
O
P
PhO
OPh
N3
R
R
O
NEt3
O
P
O
OPh
OPh
N3
R
N3
47
44
O
48
R
H
N
O
C
O
R'
43
Scheme 9: Possible mechanism for the formation of acyl azide using DPPA.
There is the possibility that side products are formed when DPPA is being used in the Curtius
rearrangement. In some cases, hydrazoic acid formed in the reaction mixture can react with the
isocyanate to give a carbamoyl azide. Urea and ester derivatives can be formed, though in rare
cases, by addition of the starting carboxylic acid to the isocyanate (Scheme 10).
RCON3
heat
O
RNH2
RNCO
N3H
RCO2H
R'OH
O
R
N
H
R
O
R
N3
O
R
N
H
O
R
N
H
N
H
N
H
R
O
O
R
Scheme 10: Possible side products in modified curtius rearrangement.[56]
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35
Recently, Lebel et al.[53] reported a one-pot synthesis of Boc-protected amines via Curtius
rearrangement using di-tert-butyl dicarbonate, sodium azide, tetrabutylammonium bromide
and zinc(II) triflate (Scheme 11).
Boc2O, NaN3
n-Bu4NBr (15 mol%)
O
R
OH
Zn(OTf)2 (3.3 mol%)
o
THF, 40-45 C
R
H
N
OtBu
O
R = aliphatic
Scheme 11: One-pot synthesis of Boc-protected amines.
In the above reaction, an excess of NaN3 is used to produce in situ BocN3 in the presence of
phase transfer catalyst n-Bu4NBr. Upon the addition of 1-adamantancarboxylic acid to this
mixture the formation of corresponding azide was observed. When the reaction mixture was
heated to 80 oC, the Curtius rearrangement occurred and gave the desired carbamate. The acyl
azide was converted to the corresponding isocyanate at 40 oC, but the carbamate was obtained
in low yield. This observation suggested that the addition of tert-butoxy moiety to the
isocyanate intermediate was slow at 40 oC. It was thought that the addition of Lewis acid in
catalytic amounts can accelerate the formation of the carbamate. After examining several
Lewis acids it was found that Zn(OTf)2 can act as very good Lewis acid catalyst for the above
transformation at 40 oC which gave more than 90% yield. This study reveals that the Zn
catalyst is not involved in the formation of the isocyanate, but rather accelerates the formation
of the carbamate through the formation of a zinc carbamoyl bromide species. The exact
kinetics and mechanism of this reaction are still unknown.
2.5.4 Ireland-Claisen Rearrangement
The Ireland-Claisen rearrangement is a mild variant of the Claisen rearrangement,
employing the allyl ester of a carboxylic acid ester instead of an allyl vinyl ether (Scheme 12).
The ester is converted to its silyl ketene acetal which rearranges at temperatures below 100 oC.
The intermediate product of the rearrangement, a carboxylic acid silyl ester can not be isolated
36
Literature Review
and is hydrolyzed during the workup. Thus, Ireland-Claisen rearrangement offers a simple
access to chain extended carboxylic acids (1,4-unsaturated carboxylic acids).
O
LDA,
TMS-Cl
O
O
OH
OSiMe3
OSiMe3
[3,3]-sigmatropic
rearrangement
O
O
H
Scheme 12: Ireland-Claisen rearrangement.
Since its introduction in 1972, the Ireland-Claisen rearrangement[59] has became widely used
in the synthesis of a diverse range of natural products and other targets.[60] The popularity of
the reaction is due to several factors: (i) the ease of preparation of the allylic ester reactants;
(ii) the ability to control the E/Z geometry of the ester enolate and hence the relative
stereochemistry between C-2 and C-3 of the pentenoic acid product; (iii) the frequently high
transfer between the allylic stereo center C-5 of the allyl ketene acetal and newly formed
stereocenters at C-2 and/or C-3 of the pentenoic acid (Scheme 13); (iv) the generally high
level of alkene stereocontrol.[61]
OSiMe3
R2
O
2
R1 5
H
OSiMe3
R2
O
2
R3
heat
3 R3
R2
R1
O
Me3SiO
R1 5
3 R3
Z-enolate
R2
OSiMe3
heat
O
R1
R2
R3
H
Me3SiO
O
R3
R1
O
R1
OSiMe3
R2
R3
E-enolate
Scheme 13: Chair-like transition states in Ireland-Claisen rearrangement.
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37
2.5.4.1 Effect of reaction conditions on the stereoselectivity of silyl ketene acetal
formation
Silyl ketene acetal geometry is controlled by the selective formation of the E- and Zester enolates. Normally esters tend to form E-enolates with bases like LDA in THF at -78 oC
by avoiding the severe 1,3 diaxial interactions between the N-isopropyl group of LDA and the
substituent in the ester as shown in the six-membered transition state (Figure 2.5.6).
1,3 axial interaction
not favored
R1
N Li O
H
H
TS I
OLi
R1
favored in case of esters
relatively low
N Li O 1,2 torsional strain
H
R1 OR
TS II
H
OR
OR
Z-enolate
OLi
OR
R1
E-enolate
Figure 2.5.6: Transition state diagram for the enolate formation of esters with LDA.
According to the transition state diagram (Figure 2.5.6), in THF solution the metal cation Li+
coordinates with the carbonyl carbon and the base. The kinetic enolization of esters in THF is
postulated to operate through a cyclic transition state TS I (Figure 2.5.6). In 1991 Ireland et
al.[62] reported that the addition of dipolar solvents (additives) such as HMPA, DMPU, or
TMEDA can switch the enolate geometry to Z, favoring the thermodynamic product of the
reaction. A switch from a preference of TS I in THF to TS II for the deprotonation in the
presence of dipolar solvents appears questionable at first, due to a severe 1,3-diaxial
interaction between the N-isopropyl group and R1 substituent in TS II. However, the presence
of additives such as HMPA or DMPU results in greater degree of solvation of the lithium
cation and weakened Li+-carbonyl oxygen interaction. A decrease in polarization of the
carbonyl oxygen bond also results in a significantly less reactant like transition states, as the αC-H bond becomes more difficult to extend and break. It is important to note that a strong
38
Literature Review
solvation of Li+ ions may lead to a relative stabilization TS II over TS I through the
occurrence of a late transition state. In a very strongly complexing solvent system, a continual
change from an expanded to cyclic to an acyclic transition state is expected. In fact, an acyclic
transition state can be considered as an extreme situation of an expanded cyclic transition state
with a strongly solvated base counter ion (Figure 2.5.7).[62, 63]
P
P
O
N
O
Li
N
P
O
Li
NMe2
Me2N
OEt
P
P
Me2N
O P
P
O
O
O
O
O
HMPA
C O can not replace the Li-HMPA bond since lithium is
very strongly coordinated to oxygens of HMPA
OEt
P
O
N
Li
O
O
H
EtO
P
P
O
H
P
EtO
CH3
O
CH3
O
O
Li
OLi
P
O
P
OEt
Z-enolate
H
no preliminary interaction between reagent and
substrate before bond reorganization (loose or late
transition state)
Figure 2.5.7: Model for solvation of lithium with HMPA and deprotonation of an ester.
Table 2 summarizes the effect of polar solvents on the formation of E/Z enolates of ethyl
propionate. After examining different combinations of solvent systems, Ireland et al. reported
that THF/45% DMPU or THF/23% HMPA are suitable solvent system for the formation of
thermodynamic Z-enolate. This proves that enolate formation can proceed in both ways either
under kinetic or thermodynamic control by changing the solvent system and thus controls the
relative stereochemistry of newly forming chiral centers. But there is no comprehensive
explanation for the formation of Z-enolate caused by the addition of dipolar solvents such as
DMPU or HMPA until today.
Literature Review
39
Table 2. Effect of solvent on stereoselectvity of silyl ketene acetal formation of ethyl
propionate with LDA
Entry
Solvent
Ester:base
Z:E
Yield%
1
THF
1:1
6:94
90
2
THF/25%TMEDA
1:1
60:40
50
3
THF/50%TMEDA
1:1
----
0
4
THF/15% DMPU
1:1
37:63
90
5
THF/30% DMPU
1:1
67:30
85
6
THF/45% DMPU
1:1
93:7
90
7
THF/23% HMPA
1:1
85:15
90
2.5.5 Syn selective Evans Aldol reaction
The aldol reaction is one of the most important methods of forming carbon-carbon
bonds. The addition of an enolate to an aldehyde leads to the formation of at least one chiral
center. This reaction, a classical method for the construction of carbon chains with oxygen
functionality in 1,3-positions, has undergone remarkable changes in the last twenty years. The
impulse for this development was given by the increasingly ambitious synthetic goals, which
were provided in particular by the macrolide and polyether antibiotics with their many
functional groups. New and particularly stereoselective variants of the aldol reaction have
proved to provide the key to success. Chiral auxiliary chemistry has been exploited in
asymmetric aldol reactions to generate both syn and anti selective products till to date since it
was pioneered by Evans et al. in 1981.[46]
In general, E-enolates of ketones or ester derivatives produce anti aldol products and Zenolates produce syn aldol reactions according to chair-like transition state proposed by
Zimmerman, well known as Zimmerman-Traxler model (Scheme 14).[64]
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Literature Review
O
OM
R2
R1
3
R
H
1
H R
R3
H
H
E enolate
R
R3
2
O
O
3
OH
R1
R
R
OM
R2
M
O
2
Z enolate
R1
O
syn aldol
product
1
H R
3
2R
R
H
O
M
O
O
OH
R1
R3
2
R
anti aldol
product
Scheme 14: Zimmerman-Traxler transition states for E- and Z enolates.
Evans et al. reported the application of chiral N-acyl oxazolidinones for diastereoselective syn
aldol reactions.[65] After pioneering, this strategy became a very important tool in the synthesis
of a broad range of bioactive natural products. Since acylated oxazolidinone is an imide, it
forms exclusively Z-enolate (Figure 2.5.1) resulting in syn aldol product. To generate the Zenolate of N-acyl oxazolidinone, the combination of dibutylboron triflate and triethylamine
were used since boron forms a strong and short bond with oxygen and thus forms a tight six
membered chair like transition state that leads to syn aldol adduct with high preference. The
observed high diastereoselectivity of one syn product over the other is due to the arrangement
of the carbonyl group of the oxazolidinone and the C-O bond of enolate arranges in an anti
fashion to each other in order to minimize dipole repulsions. In this arrangement the aldehyde
approaches the enolate from the less hindered side of the chiral auxiliary (Scheme 15).
Literature Review
41
O
O
O
O
N
O
OH
N
RCHO
H
H
Me
49
R
B
O
50
O
B
Xc
Me
H
O
R
O
51 "Evans" syn
O
N
O
N
Me
O
R
O
O
O
H
H
Xc
H
R
Me
OH
O
OH
Me
R
Other view
Scheme 15: Favored transition states in the asymmetric aldol addition of boron enolates of
chiral N-acyl oxazolidinones.
More recently, Crimmins and coworkers published a detailed account of their work in
asymmetric aldol additions employing titanium(IV) enolates of N-acyloxazolidinones, Nacyloxazolidinethiones and N-acylthiazolidinethiones.[66] Crimmins addressed the importance
of the dialkylboron enolates of N-acyloxazolidinones as the most commonly used enolates for
the preparation of the Evans syn products.
The reaction with boron enolates proceeds via the non-chelated transition state 54 to deliver
the well-known Evans syn products 55 (Scheme 16). However, the use of titanium(IV)
enolates of N-acyloxazolidinethiones and N-acylthiazolidinethiones allows the reaction to
proceed via the chelated transition state 56 to deliver the non-Evans syn products 60.
Furthermore, Crimmins reported the potential of titanium(IV) enolates of N-acyloxazolidinone
24, N-acyloxazolidinethiones 52 and N-acylthiazolidinethiones 53 for the preparation of both
Evans and non-Evans syn aldol products by variation of the reaction conditions.
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Literature Review
X
Y
Bn
N H
LxM
O
R
O
Y
X
O
N
R
N
H
Bn
55
"Evans" syn
54
Bn
X
Me
Me
RCHO
Y
OH O
X
24 X = O, Y = O (Oxazolidinone)
52 X = O, Y = S (Oxazolidinethione)
53 X = S, Y = S (Thiazolidinethione)
Bn
H
H
R
Y
N
O
O
Cl
Ti Cl
Cl
OH
Y
O
R
N
X
Me
Bn
Me
56
57
"non-Evans" syn
Scheme 17: Non-chelated (54) and chelated (56) transition states in the asymmetric aldol
addition of titanium(IV) enolates of N-acyloxazolidinone, N-acyloxazolidinethiones and
thiazolidinethiones.
Crimmins found that the diastereoselectivity of the titanium(IV) enolates of Nacyloxazolidinone, N-acyloxazolidinethiones and N-acylthiazolidinethiones to deliver the
Evans syn product 55 is dependent on the nature and amount of the base used to generate the
enolates. The Evans syn products were obtained, via the non-chelated transition state 54, when
titanium enolates were formed in the presence of two equivalents of a base such as (–)sparteine. It was suggested that the second equivalent of amine coordinates to the metal center
preventing further coordination of the imide or thioimide carbonyl to the metal center. NonEvans syn products were obtained when only one equivalent of amine was used to generate the
enolates of N-acyloxazolidinethiones and N-acylthiazolidinethiones. In this case the imide
carbonyl or the thiocarbonyl coordinated to the metal center to produce the highly ordered
chelated transition state 56. Coordination of the imide carbonyl or thiocarbonyl to the metal
center led to reversal of the π-facial orientation of the enolate in the transition state.[66]
Literature Review
43
2.5.6 Esterification using the Yamaguchi method and DCC/DMAP conditions
2.5.6.1 Yamaguchi esterification
Yamaguchi esterification[67] is one of the important tools in the total synthesis of many
biological active natural and unnatural lactones as they contain at least one ester bond in their
core structure. The Yamaguchi esterification allows mild conditions for the synthesis of highly
functionalized esters.[68-71] The reaction sequence involves the formation of mixed anhydride
60 using the so-called Yamaguchi reagent i.e 2,4,6-trichlorobenzoyl chloride 59 with the
carboxylic acid. The reaction of an alcohol with the mixed anhydride 60 in presence of DMAP
generates the desired ester (Scheme 17).
Cl
O
Cl
O
R
Cl
OH
59
Cl
O
R
O
Cl
O
DMAP, toluene
Et3N, CH2Cl2
58
O
R'OH
Cl
Cl
R
O
R'
61
60
N
N
DMAP
64
Scheme 17: Yamaguchi esterification.
The mechanism involves the replacement of the chloride in 2,4,6-trichlorobenzoyl chloride by
the carboxylate giving rise to mixed anhydride 60. DMAP (64) is a stronger nucleophile than
alcohol, therefore it attacks the mixed anhydride 60. Since the chlorine atoms on the benzene
ring create steric crowding around the aromatic carbonyl group in the mixed anhydride 60,
DMAP attacks regioselectively on the carbonyl group of the former carboxylic acid leading to
the formation of an acylated pyridinium salt 62 between carboxylic acid and DMAP. As
DMAP is a good leaving group, attack of alcohol is quite fast and leads to ester bond
formation (Scheme 18).
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Literature Review
Cl
O
O
Cl
R
R
Cl
59
N
O
R
O
H
O
Cl
N
Cl
N
Cl
60
H
NEt3
N
O
O
Cl
O
Cl
Cl
O
O
R'
Cl 63
62
steric hindrance of chloro
substituents blocks attack of
this carbonyl group
O
R
O
R'
N
61
N
DMAP
Scheme 18: Mechanismof the Yamaguchi esterification.
Recently Santalucia Jr. et al.[72] showed that aliphatic carboxylic acids can form ester bonds in
a single step procedure by reacting with para-substituted unhindered benzoyl chlorides through
the intermediacy of aliphatic symmetrical anhydrides. The byproduct of this step is the
aliphatic carboxylate, which reenters the cycle (Scheme 19). Thus, until the regioselective
completion of the reaction, there is always aliphatic carboxylate remaining, competing with
the aromatic carboxylate, and the alcohol. The mechanism is based on the assumption that
aliphatic carboxylates are better nucleophiles than aromatic carboxylates and alcohols.
However, the aliphatic anhydride is also more electrophilic towards DMAP (not shown in the
Scheme) or the alcohol than is the aromatic carbonyl of the mixed anhydride that is produced
in situ. This proposed mechanism suggests that any aromatic acid chloride should be capable
of producing preferentially and in situ the symmetric aliphatic anhydrides that then could be
used in regioselective synthesis of aliphatic esters. It is important to consider the relationship
between steric effects, electronic effects, and reactivity. The aliphatic anhydride produced in
situ must be more electrophilic toward the alcohol than the aromatic carbonyl of the mixed
aliphatic-aromatic anhydride for this procedure to succeed. p-Toluoyl chloride proved to be
ideal for this transformation and catalytic amounts of DMAP are sufficient for the efficient
transformation.
Literature Review
45
O
R
O
Ar
O
O
Ar
O
OH
R
O
Cl
Ar
OH
O
R
O
O
R
O
O
R
Ar = p-toluoyl
O
R
O
R'
R'OH
Scheme 19: Proposed reaction cycle for the esterification using aromatic acid chlorides.
2.5.6.2 Esterification using DCC/DMAP conditions (Steglich esterification)
Esterification using dicyclohexylcarbodiimide (DCC) 65 is a mild reaction which
allows the conversion of sterically demanding and acid labile substrates.[73, 74] It is one of the
convenient methods for the formation of tert-butyl esters because t-BuOH tends to form
carbocations and isobutene in acidic conditions.
N
C
N DCC
65
Carboxylic acid reacts with DCC 65 to form O-acylisourea 66 intermediate, which offers
similar reactivity to the corresponding acid anhydride like in the Yamaguchi esterification.
Now the alcohol may attack the activated carboxylic acid to form an ester and stable
dicyclohexyl urea 67 (DHU). But the attack of the alcohol is slower due to less nucleophilicity
of the alcohol towards the O-acyl isourea 66. Without the presence of catalytic amounts of bi
character species (nucleophile as well as leaving group) such as DMAP, the acyl group can
migrate to the nitrogen, leading to the formation of N-acyl-dicyclohexyl urea 68. A common
explanation of the DMAP acceleration suggests that DMAP, as a stronger nucleophile than
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Literature Review
alcohol, reacts with the O-acylisourea 66 leading to a reactive acyl pyridinium species 62
("active ester"). This intermediate cannot form intramolecular side products but reacts rapidly
with alcohols. DMAP acts as an acyl transfer reagent in this way, and subsequent reaction with
the alcohol gives the ester (Scheme 20).
O
R
O
O
O
H
N
R
R
O
C N
NH
O
C
N
HN
C
N
66
65
O
R
O
C N
NH
O
H
O
R'
HN
R
O
N
H
67
66
O
R
O
C N
NH
N
H
68
66
O
C
O
H
O
R'
R
O
R'
O
N
R
O
R
O
C N
NH
N
O
N
R
HN
N
H
67
O
N
N
O
H
O
R'
62
Scheme 20: Mechanism for the formation of ester using DCC 62.
R
O
R'
Literature Review
47
2.5.7 Coupling reagents in solution phase peptide synthesis
A large number of natural products are based upon a peptide framework and exhibit a
spectrum of biological activity. Over 200 new peptide based drugs are under different stages
of development with 50% of them under clinical trials are prior to approval.[75] Due to the
importance of peptides, it has become a challenge for synthetic chemists to develop
methodologies to construct peptides over the past few decades.[76, 77] A key step in the peptide
production process is the formation of an amide bond. This requires activation of carboxylic
acids which is usually carried out using so-called the ‘Peptide coupling reagents’ (Scheme
21).
R''
R'
R
N
H
OH
O
Coupling reagent
R
N
H
OR'''
H2N
R'
X
O
O
X = activating group
R'
R
N
H
O
H
N
O
OR'''
R''
Scheme 21: General strategy for peptide bond formation.
The area of coupling reagents began in the early 1950’s with the introduction of DCC 65,
which at that time was already known and well studied. The difficulty in the complete removal
of dicyclohexyl urea 67, the byproduct from the reaction mixture, led to the development of
several modified carbodiimide reagents (Figure 2.5.9).
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Literature Review
H3 C
CH3
CH3
N C N
N C N
H3C
N C N
H3C
N
H3C
DCC (65)
urea formed is partially soluble
in many solvents and hard to
purify via column chromatography
CH3
EDC (69)
water soluble by-product is
easily removed in aqueous
work-up
urea formed is soluble in most
organic solvents. This is advanta-geous in solid phase synthesis
CH3
N C N
CH3
N C N
N C N
N C N
H3 C
H3C CH3
BEC (73)
CIC (71)
DIC (70)
H3C
H3C CH3
BMC (74)
N,N'-dicyclopentyl
carbodiimide (72)
Figure 2.5.9: Various carbodiimide coupling reagents[78]
Unfortunately, carbodiimides did not come close to being ‘ultimate’ coupling reagents,
because their high reactivity provokes racemization and side reactions. As shown in the
esterification with DCC (Scheme 20), N-protected amino acid reacts with carbodiimide to
form O-acyl isourea 75 which is the active species for the amide bond formation (Scheme 22).
The O-acyl isourea 75 can react with the free amino group of an amino acid to give the
corresponding amide 76. This is the desired way of the process. The reactive O-acyl isourea
75, however, can undergo enolization to 77 with a corresponding loss of chirality.
Literature Review
49
R
Pg
O
H
N
N C N
R
OH
R1
N-protected amino acid
Pg
N
O
O
O
N
O
R1
R
Pg
H2N
O
O
N
H
R1
O
R2
PG
O
76 peptide
PG
O-protected amino
acid
R
O
N
H
N
R
Pg
O
N
H
R1
O-acyl isourea
O
N
N
H
R
N-acyl urea 78
R
R
N
R
N
H
Pg
O
H
N
enolisation leads
to recemic product
R
N
H
77
H
N
N
H
O
H
N
R2
enolisation
OH
R
N
O
R1
75
Pg = protecting
group
H
N
Pg
O
H
N
R
R1
slow
O
O
oxazolone 79
tautomerization
O
R2
N
H
O
Pg
O
peptide
PG
R2
R1
O
80
R1
O
H2 N
N
Pg
O
H
N
loss of chirality
OH
Scheme 22: Activation of N-protected amino acids with carbodiimide reagents.
Furthermore, it can also suffer a rearrangement to give the N-acyl urea 78, which is not
reactive. Last, but not least, it can sustain an intramolecular cyclization to give 5(4H)oxazolone 79, which is less reactive than the O-acyl isourea and can tautomerize to 80 with a
corresponding loss of chirality as well.
At the beginning of the 1970’s 1-hydroxy benzotriazole 81 (HOBt) was proposed as additive
to DCC 65 to suppress racemization during the peptide coupling.[79,
80]
Since then other
benzotriazole derivatives such as 1-hydroxy-5-chloro-benzotriazole 82 (Cl-HOBt) or 1-
50
Literature Review
hydroxy-7-aza-benzotriazole 83 have also been used. The OBt-active esters of amino acids are
less reactive than the O-acyl isourea, but more stable and less prone to racemization. All these
factors make the addition of benzotriazole derivatives almost mandatory to maintain high
yields and chiral configuration during the peptide bond formation with carbodiimide activation
(Scheme 23).
GP
O
H
N
N
O
R
N
H
R1
R
GP
O
H
N
O
N N
N
GP
R1
HO
N
N
N
O
H
N
R1
O
H2N
O
R2
O
N
H
PG
O
peptide
PG
R2
N
N
N
N
N
OH
N
N
OH
Cl
HOBt 81
N
N
N
OH
HOAt 83
Cl-HOBt 82
Scheme 23: Formation of peptide bonds via OBt esters.
In the last decade onium (phosphonium and ammonium/uronium) salts of hydroxybenzotriazole derivatives have been introduced for the formation of peptide bonds.[77]
N
O
O
N
N
N PF6
H3C
CH3
N
N
CH3 CH3
HATU 84
N
N
N PF6
H3C
CH3
N
N
CH3 CH3
HBTU 85
Cl
H3C
O
N
N
N BF4
O
N
N
N PF6
CH3
N
N
CH3 CH3
H3C
HCTU 86
CH3
N
N
CH3 CH3
TBTU 87
Figure 2.5.10: Common uronium coupling reagents.
HATU = N-[Dimethylamino)-1H-1,2,3-triazolo[4,5-b]pyridine-1-methylene]-N-methaminium
hexafluorophosphate; HBTU = N-[(1H-benzotriazolo-1-yl)(dimethylamino)methylene]-N-
Literature Review
51
methylmethanaminium hexafluorphosphate N-oxide; HCTU = N-[(1H-6-Chlorobenzotriazolo1-yl)(dimethylamino)methylene]-N-methylmethanaminium hexafluorphosphate N-oxide.
The species that reacts with onium salts is the carboxylate and therefore the presence of at
least one equivalent of base is essential. The intermediate species, acyloxy-phosponium or
amidinium salts have not been detected and react immediately with the benzotriazole
derivative to give the corresponding OBt ester, which reacts with the amino component to give
the corresponding amide (Scheme 24).
O
N
N
N
O
N
N O
N
O
H3C
N R
N
H3C H3C CH3
CH3
N
CH3
H3C N CH
3
R
O
O
N
N
N
88
O
O
R
CH3
H C
O3 N
CH3
R
O
N
89 CH3
O
N N
N
90
O
R
R1
N
H
O
PG
O
O
H2N
O
PG
peptide 91
R1
Scheme 24: Mechanism for the formation of peptide using uronium reagents.
It is difficult to form a peptide bond with N-alkyl amino acids as the steric bulk of an alkyl
group on the amine reduces its nucleophilicity, slowing the reaction rate and thus leading to
undesired side products. Furthermore, racemization is more problematic because of the αproton which is now the most acidic proton whereas in natural amino acids the amide proton
would be deprotonated first. It is important to note that in the case of N-methyl amino acids,
HOBt suppresses the reaction rate and benzotriazole base reagents should be avoided in most
cases. But there are some reagents which make the N-alkyl amino acid coupling feasible.
PyBroP, PyCloP, BOP-Cl are the most general reagents for N-alkyl coupling reactions.[81]
52
Literature Review
N
N
P
X PF6
N
X=Cl, PyCloP 92
=Br, PyBroP 93
O
O
O
O
P
N
N
O
Cl
BOP-Cl 94
Figure 2.5.11: Common reagents for the peptide coupling of N-alkyl amino acids derivatives.
PyCloP = Chlorotrispyrrolidinophosphonium hexafluorophosphate;
PyBroP = Bromotrispyrrolidiniphosphonium hexafluorophosphate;
BOP-Cl = Bis(2-oxo-3-oxazolidinyl)phosphonic chloride.
Goal of research
53
3 Goal of research
The purpose of the present project is to construct conformationally constrained
analogues of the depsipeptide jasplakinolide. Constraining the conformation of a peptide
fragment by incorporating it in a macrocyclic structure represents an important strategy for
enhancing both the binding strength and selectivity. In addition, this maneuver can suppress
unwanted proteolysis. Studying the solution conformation of such a macrocyclic structure can
provide important information on the peptide surface structure and the area that is presented to
a receptor. Jasplakinolide, geodiamolide, chondramide, and doliculide display similar
biological properties. Although several syntheses of jasplakinolide had been reported when
this project began, there were no reports where variations had been performed. Jasplakinolide
is ideally suited for modification due to its modular structure. As we discussed earlier,
jasplakinolide is composed of hydrophobic polypropionate and polar peptide chains. The
interest for the construction of analogues of jasplakinolide and as well as the others in the
family of these novel biologically active macrocycles, lies in the synthesis of the
polypropionate fragment as it might function as a conformational constraining element for
these macrocycles through non bonded interactions like syn-pentane and 1,3-allylic
interactions. Even though the synthesis of the polypropionate part, a 8-hydroxy acid is
feasible,[43, 82, 83] the preparation of larger amounts is quite costly. Keeping the non bonded
interactions as conformational controlling elements in mind, novel ω-amino and hydroxy acids
were rationally designed in order to synthesize and study the solution conformations of
jasplakinolide like cyclic peptides and depsipeptides.
Two recent publications from Terracciano et al. pointed to the importance of the
polypropionate
part
in
the
construction
of
conformationally
rigid
analogues
of
jasplakinolide.[44, 45] Due to the lack of non bonded interactions in the polypropionate part of
the Terracciano analogues, there were neither good restriction in the conformations nor good
biological activity which supports our idea behind the synthesis of jasplakinolide analogues
using rationally designed amino and hydroxy acids.
54
Goal of research
The objective of this study was the design and synthesis of novel amino and hydroxy
acids which are bearing conformational controlling elements and to use them for constructing
jasplakinolide like molecules in order to understand the structure-activity relationship using
conformational and biological studies. These newly synthesized analogues should give some
idea about the pharmacophoric part of the natural product.
Results and Discussion
55
4 Results and Discussion
4.1 Design of the novel ω-amino and hydroxy acids
The design of the amino acid 95 followed from looking at the conformational control
elements in the hydroxy acid 3 (Figure 4.1.1). Thus, the 1,3-allylic interaction around the
central trisubstituted double bond should position the allylic C-H in an eclipsed orientation to
the double bond.[40] As a consequence, the methyl group at C-6 will point downwards and
orient the 2-hydroxypropyl terminus to the other side, out of the plane of the double bond. The
conformational situation at the carboxyl terminus seems to be less defined. Nevertheless,
avoidance of syn-pentane interaction between C2-Me and C4-Me will cause the carboxyl
group to point out of the plane of the central double bond as well. Our design plan then called
for a rigidification of the vinylic C5-C6 bond. Accordingly, the allylic H was replaced with a
two-carbon segment (see dashed lines in Figure 4.1.1), resulting in a meta-substituted aryl
core. This simplification removes the stereocenter at C-6.
syn-pentane interaction
1,3-allylic interaction
simplify
HO
CO2H
RHN
95 R = Boc
3
H3C
8
OH
H
4
6
H
H3C
1
CO2H
H
CH3
2
H3C
8
OH
H
4
CH3
6
H
H3C
eclipsed
H
X
H3C
H
CO2H
CH3
CO2H
1
CO2H
H
CH3
2
CH2
connect atoms to form a ring
95 X = NHBoc
Figure 4.1.1: Design of the novel ω-amino acid 95.[84]
56
Results and Discussion
In order to probe the design process, conformational search runs (Macromodel 7.0, MM2*
force field, 1000 starting structures) were carried out on hydroxy acid 3 and the amino acid 95
(without the N-Boc protecting group). The search for the hydroxy acid 3 found four
conformers within 4.184 kJ mol-1 of the minimum. The lowest conformer has the allylic C-H
eclipsing the C4-Me, but only the carboxyl group is pointing out of the plane of the double
bond (Figure 4.1.2). In the next lowest conformer 3b (ΔE = 0.93 kJ mol-1) 6-H, surprisingly, is
95a
95b
Figure 4.1.2: Calculated low energy conformations of the hydroxyacid 3 and amino acid 95
(Chem3D representations).
anti to the C4-Me. Basically, C4-Me is bisecting the angle C6-Me/C-6/C-7. This orients both
termini to the other side of the central л-system. Conformers 3c (ΔE = 2.00 kJ mol-1) and 3d
(ΔE = 4.10 kJ mol-1) actually match the expected conformation. Thus, 6-H eclipses the vinylic
methyl and both termini extend nicely to one side. For the amino acid 95 we found two
minimum energy conformers within 4.184 kJ mol-1 of the absolute minimum (E = 36.0 kJ mol1
). In all cases the termini are pointed more or less orthogonal to the plane of the aryl ring. In
Results and Discussion
57
the second lowest conformer 95b (ΔE = 2.45 kJ mol-1) the termini point to the opposite side of
the aryl ring. Conformer 95b does have striking similarity to the conformer 3c of the hydroxy
acid. Most likely electrostatic interaction and hydrogen bonding cause a substantial gain in
energy if both groups point to the same side. By looking at several of the calculated minima, it
seems that the conformation of the aryl analogue is more ordered and less flexible. An overlay
of 3c and 95b shows a decent overlap validating the original design (Figure 4.1.3).
Figure 4.1.3: Overlay of the calculated conformers 3c and 95b (grey, hydroxy acid; black,
aromatic amino acid).
Based on the non-bonded interactions the hydroxy acid 96 and the corresponding ω-amino
acid 97 were designed as well. These are truncated versions of the 8-hydroxy acid 3 of
jasplakinolide.
CO2H
RO
96 R = TBS or TBDPS
RHN
CO2H
97 R = Boc
Figure 4.1.4: Structures of hydroxy- and amino acids 96 and 97.
It can be seen that the amino and hydroxy acids 96 and 97 have one 1,3–allylic interaction and
one syn pentane interaction. Due to the non bonded interactions, the methyl groups avoid
steric repulsions and make the molecules less flexible. As a result of this, the carboxyl and
hydroxy or amino groups at the end of the chain might point in one direction, thereby allowing
bridging with a peptide fragment.
58
Results and Discussion
4.2 Retrosynthetic analysis and synthetic pathways for the designed amino
and hydroxy acids
4.2.1 Retrosynthetic analysis of amino acid 95
BocHN 7
95
1 CO2H
The amino acid 95 contains two stereocenters. If the amino function is generated by
Curtius rearrangement, a possible precursor could be the C2 symmetric dicarboxylic acid 98.
This symmetry might be exploited for the synthesis of 95 (Scheme 26).
CO2H
RHN
98
O
TBSO
CO2H
HO2C
95
Br
99
O
O
N
100 Bn
Scheme 26: Retrosynthetic plan for the novel ω-amino acid 95.
Initially, the synthetic plan was to create both stereocenters individually by using asymmetric
Evans alkylation as shown in Scheme 27. The amino group in the amino acid 95 could be
obtained from a Curtius rearrangement of the acid 101 which can be synthesized by the
hydrolysis of Evans alkylation product, which in turn could be obtained by alkylation of
propionyl oxazolidinone 100 with benzyl bromide derivative 99. As the benzyl bromide
derivative 99 is not commercially available, it needs at least 3 steps to synthesize 99. The total
sequence would require around 10 steps to synthesize the amino acid 95 excluding the
synthesis of chiral reagent 100.
Results and Discussion
O
O
alkylation
N
O
59
Bn
100
CO2H
TBSO
Br
OTBS
Curtius
rearrangement
101
99
NHBoc
TBSO
1. deprotection
2. tosylation
HO2C
NHBoc
3. iodination / alkylation
4. oxidative cleavage
102
95
Scheme 27: Initial synthetic plan for the novel ω-amino acid 95.
Based on the symmetry considerations discussed before, another route was developed for the
sythesis of amino acid 95 from commercially available dibromo benzyl derivative 104 as
shown in Scheme 28. In this pathway, amino acid 95 could be obtained by Curtius
rearrangement and hydrolysis of the mono acid 103, which can be obtained from the diacid 98
either by mono esterification in one step or by diesterification followed by selective enzymatic
monohydrolysis in two steps. The diacid 98 can be obtained in two steps by double alkylation
using propionyl oxazolidinone 100 with dibromo benzyl derivative 104 followed by hydrolytic
removal of the chiral auxiliary.[85]
HO2C
NHBoc 1. Curtius
CO2H
MeO2C
1. diesterification
2. Enzymatic mono
hydrolysis
2. hydrolysis
95
103
CO2H
HO2C
98
1. alkylation
2. oxidative
cleavage
Br
1. mono esterification
or
Br
104
Scheme 28: Alternative pathway for amino acid 95.
60
Results and Discussion
4.2.2 Synthesis of amino acid 95
The synthesis of amino acid 95 began with commercially available 1,3bis(bromomethyl)benzene (104), which was subjected to double alkylation with propionyl
oxazolidinone 100. The propionyl oxazolidinone 100 was synthesized from R-phenylalanine
105 according to an Evans procedure[86, 87] as shown in Scheme 29.
O
BF3.OEt2
BH3.SMe2
NH2
OH
105
O
n-BuLi
O
H3C
(EtO)2CO
K2CO3
NH2
OH
THF, reflux
90%
89%
106
THF, -78oC
93%
Bn
O
107
O
O
Cl
HN
N
O
Bn
100
Scheme 29: Synthesis of N-propionyl-oxazolidinone 100.
Initially for the alkylation, NaHDMS was used as a base. Even though the dialkylated product
was formed, the yield was very low irrespective of the amount of excess chiral reagent 100
used and the mono alkylated product formed predominantly. LDA proved to be good reagent
for this dialkylation. In small scale reaction yields were moderate varying from 50-55% and in
large scale reaction yields were better producing up to 60% of the desired compound 108.
(Scheme 30). The yield might be increased by using excess of propionyl oxazolidinone.
Oxidative hydrolysis of chiral auxiliary led to the diacid 98, which was in turn converted to the
dimethyl ester 109 via DCC-mediated esterification. This maneuver was necessary in order to
allow for a monohydrolysis of the homotopic ester groups. This could be achieved by esterase
induced hydrolysis. It is necessary to use only catalytic amounts of pig liver esterase for this
purpose. Otherwise, over hydrolysis can occur in no time and convert diester 109 back to the
diacid 98. In this case, 100 units (2.5 mg) of pig liver esterase were used for the conversion of
Results and Discussion
61
one mmol of dimethyl ester 109 to mono ester 103 in 12 h at room temperature (Scheme
30).[88]
O
O
N
O
O
Br
Br
LDA, THF
O
-78 oC to rt
58%
Bn
100
O
N
N
Bn
O
O
HO
MeOH, DCC,
DMAP
OH
THF, 23 oC
95%
CH2Cl2, 23 oC
71%
98
O
O
MeO
23 oC, 64%
O
O
PLE, H2O, pH 7.5
OMe
O
Bn
108
104
30 wt% H2O2,
LiOH in H2O
O
O
MeO
OH
103
109
Scheme 30: Synthesis of mono ester 105.
With the monoester 103 in hand, the Curtius rearrangement was tried to synthesize the Fmoc
protected amino acid in presence of DPPA/NEt3 and 9H-fluoren-9-ylmethanol (Scheme 31).
However, the reaction did not work and the reactant got decomposed. The same was observed
when benzyl alcohol was used in order to obtain the Cbz protected amino acid as reported in
literature.[89] Also attempts failed to synthesize the unprotected amine by Curtius
rearrangement using 1 N HCl as reported in our lab.[90] Then it was decided to follow the same
procedure as reported in the original literature[57,
58]
to make the Boc protected amino acid
using t-BuOH as the nucleophile for attack on the isocyanate 110. Initially the reaction worked
with very low yield and the major product obtained was the urea derivative 114. The reason
for obtaining the urea 114 might be due to water in t-BuOH. Then some isocyanate could react
with water and form the amine 113, which in turn would act as nucleophile and attack the
isocyanate as shown in Scheme 32. This result was confirmed by using NMR and HRMS
methods. As both products were not separable by column chromatography the NMR was not
very pure.
62
Results and Discussion
O
O
MeO
O
O
OH
DPPA, NEt3
toluene, reflux,
3h
103
C
N
MeO
110
9H-fluoren 9ylmethanol
O
NHFmoc
MeO
reflux
O
benzyl alcohol
111
NHCbz
MeO
reflux
O
112
1N HCl, reflux
NH2
MeO
113
Scheme 31: Attempts to perform the Curtius rearrangement on acid 103.
O
O
C
N
MeO
OMe
H2N
O
O
MeO
H
N
H
N
O
OMe
O
114
Scheme 32: Mechanism for the formation of the urea 114.
After realizing that tert-butanol can act as good nucleophile towards isocyanate 110, the mono
acid 103 was treated with diphenylphosporyl azide followed by heating of the reaction mixture
in the presence of distilled tert-butanol. This affected a Curtius rearrangement resulting in the
Results and Discussion
63
N-Boc protected ω-amino acid ester 115 in good yield. Basic hydrolysis of the ester led to the
desired acid 95 (Scheme 33). This route secures the novel amino acid 95 in gram quantities.
O
O
MeO
C
DPPA, NEt3
OH
toluene, reflux
3h
N
MeO
110
103
O
tBuOH
reflux
72%
O
O
NHBoc
MeO
NaOH
THF/H2O
80%
95
115
Scheme 33: Synthesis of amino acid 95
After successfully achieving the synthesis of ω-amino acid 95, we decided to synthesize the
corresponding bromo amino acid 116 which has the bromine in the 3rd position of the aromatic
ring of the amino acid 95. The purpose of the synthesis amino acid 116 is, that it can create
conformational constraint in cyclic peptides and at the same time some side chains could be
attatched to the resulting peptides through the aromatic region of this amino acid using various
coupling reactions.
Br
BocHN
CO2H
116
Almost the same strategy was used in order to synthesize bromo amino acid 116 (cf. Schemes
30 and 33). The synthesis of amino acid 116 started with commericially available 3,5dimethylbromobenzene 117. Radical bromination of 117 using N-bromosuccinimide in
presence of AIBN provided the tribromo benzyl derivarive 118 in 30% yield.[91, 92] Alkylation
of propionyl oxazolidinone 100 with bromo benzyl derivative 118 using LDA at -78 oC
produced the double alkylated product 119 in 53% yield (Scheme 34). Subsequent cleavage of
the chiral auxiliary using mild oxidative hydrolysis conditions (H2O2 / LiOH) gave the diacid
64
Results and Discussion
120 in 89% yield. DCC mediated esterification of diacid 120 with methanol in presence of
catalytic amounts of DMAP produced the dimethyl ester 121 in 75% yield. Enzymatic
monohydrolysis of the diester 121 using Pig liver esterase led to the monoester 122 in 57%
yield. Curtius rearrangement of the resulting monoester 122 using DPPA in presence of
triethylamine in t-BuOH afforded the corresponding Boc protected amino acid methylester
123 in 60% yield. Basic hydrolysis of the methylester 123 using 0.4 N NaOH produced the
desired bromo amino acid 116 in 80% yield.
Br
NBS, AIBN
Br
O
N
N
O
Bn
Bn
-78 oC to rt
53%
100
30 wt% H2O2,
LiOH in H2O
O
O
O
O
HO
THF, 23 oC
89%
OH
Br
120
Br
119
MeOH, DCC,
DMAP
CH2Cl2, 23 oC
75%
LDA, THF
O
Bn
118
117
O
Br
N
CCl4, reflux, 20 h
30%
Br
O
O
O
CH3
H3C
O
O
PLE, H2O, pH 7.5
MeO
OMe
23 oC, 57%
Br
121
O
O
O
HO
DPPA, NEt3
OMe
Br
123
Br
122
0.4 N NaOH, THF, H2O
rt, 2 h, 80%
t-BuOH, reflux
60%
NHBoc
MeO
116
Scheme 34: Synthesis of bromo amino acid 116.
Results and Discussion
65
4.2.3 Synthesis of jasplakinolide analogues by incorporating the amino acid 95
With the amino acid 95 in hand, the synthesis of various macrocyles was targeted, in
which the two ends of the acid 95 are bridged with some tripeptide fragments. Our goal was to
prepare pairs of tripeptide fragments with one of them carrying a N-methyl group at the middle
amino acid. The conformational analysis of the corresponding macrocycles should provide
some hints on the mutual influence of the parts contained in the macrocycle. For this study Lphenylalanine, D-alanine, L-alanine were chosen to form a tripeptide. The tripeptide was
assembled by a classical Boc strategy as shown in Scheme 35. That is, DCC-mediated
condensation of the phenylalanine derivative 124 with N-Boc-D-alanine 125 gave the dipetide
126. After cleavage of the Boc protecting group with trifluoroacetic acid, coupling of the free
amine with N-Boc-L-alanine using the coupling reagent 1-ethyl-3-(3-dimethylaminopropyl)
carbodiimide hydrochloride (EDCI) provided the tripeptide 127 in 75% yield. Cleavage of Boc
using TFA and TBTU mediated condensation of the resulting amine with the amino acid 95
led to the acyclic tetrapeptide 128. Hydrolysis of the methyl ester, removal of the Boc group
and macrolactam formation with 2-(1H-benzotriazole-1-yl)-1,1,3,3-tetramethyluronium
tetrafluoroborate (TBTU) in DMF (0.001 M) gave rise to jasplakinolide (geodiamolide)
analogue 129 in 50% yield (Scheme 35).
66
Results and Discussion
Ph
Ph
Me
CO2Me
H2N
H
124
N
CO2H
DCC, HOBt
Boc
THF, 0-23 C
80%
H
o
O
H
125
CO2Me
N
N
Boc
126
Ph
H
1. TFA, CH2Cl2, 23 oC
2. EDCl, HOBt, Boc-L-Ala-OH,
Et3N, THF/CH2Cl2 5:1, 23 oC
75%
Me
H
N
CO2Me
N
1. TFA, CH2Cl2, 23 oC
O
O
N
H
127
Boc
2. TBTU, HOBt, amino acid 95,
iPr2NEt, DMF, 23 oC
75%
Ph
Ph
H
Me
H
N
CO2Me
N
O
O
N
NHBoc
1. NaOH, THF/H2O, 23 oC
2. TFA, CH2Cl2, 23 oC
3. TBTU, HOBt, iPr2NEt,
DMF, 23 oC, 50% (3 steps)
H
O
128
H
Me
O
N
N
N
O
O
Me
N
H
H
H
O
129
Scheme 35: Synthesis of jasplakinolide (geodiamolide) analogue 129.
In a similar manner, tripeptide 132 was assembled (Scheme 36). Here, N-Boc-N-methyl-Dalanine[93, 94] 130 became the central amino acid fragment. For the formation of the peptide
bond to the N-methylated amine, the coupling reagent bromotrispyrrolidinophosphonium
hexafluorophosphate (PyBroP) came to use. After liberation of the terminal amine using TFA,
EDCI-mediated condensation with the amino acid 95 provided the seco-compound 133. Ester
hydrolysis, cleavage of the Boc protecting group and macrolactam formation using the same
conditions as for 129 delivered the jasplakinolide (geodiamolide) analogue 134 with a Nmethyl amide group.
Results and Discussion
67
Ph
Ph
Me
CO2Me
H2N
Me
124
N
CO2H
DCC, HOBt
Boc
THF, 0-23 oC
70%
H
CO2Me
N
O
Me
130
N
Boc
131
Ph
H
o
1. TFA, CH2Cl2, 23 C
Me
2. PyBroP, iPr2NEt,
N-Boc-L-Ala-OH,
CH2Cl2, 23oC
60%
Me
Me
Me
N
1. TFA, CH2Cl2, 23 oC
O
O
N
H
Boc
2. EDCl, HOBt, amino acid 95,
iPr2NEt, THF/CH2Cl2 5:1,
23 oC, 52%
132
Ph
H
N
CO2Me
N
CO2Me
N
O
O
N
O
NHBoc
1. NaOH, THF/H2O, 23 oC
2. TFA, CH2Cl2, 23 oC
Ph
H
Me
N
3. TBTU, HOBt, iPr2NEt,
Me
o
DMF, 23 C, 50%(3 steps)
Me
H
133
O
N
N
H
O
O
N
O
H
134
Scheme 36: Synthesis of jasplakinolide (geodiamolide) analogue 134 containing a N-methyl
amide bond.
One more pair of jasplakinolide analogues 135 and 136 were synthesized by incorporating the
amino acid 95. These analogues were prepared using a similar strategy by Shazia Yasmeen.
The variation of these analogues from the others 129 and 134 is the presence of a β-amino
acid. The compounds 135 and 136 are structurally similar to jasplakinolide as both are 19membered macrocycles. The four analogues containing the amino acid 95 were synthesized in
order to probe the conformational variations between the substrates containing different ring
size and effect of N-methyl amide bond on the conformations of similar macrocycles.[84] The
detailed conformational analysis will be discussed in the conformational analysis section.
68
Results and Discussion
OMe
OMe
O
O
H
H
N
N
N
N
O
O
Me
N
H
H
H
H
N
N
O
O
Me
N
Me
H
N
N
H
H
O
O
136
135
Figure 4.2.1: Jasplakinolide analogues containing a β-amino acid and amino acid 95.
4.2.4 Retrosynthetic analysis of hydoxy and amino acids 96 and 97
While the amino acid 95 was relatively easy to synthesize, it seemed that the aryl ring
imposes too much conformational constraint. Therefore we decided to design and synthesize
truncated versions of the hydroxy acid 3 of jasplakinolide. By removing a syn-pentane
interaction on the alcohol terminus we reached hydroxy acid 96 and amino acid 97. They
should be easily accessible. One further advantage would be that the pair 96/97 should allow
to probe the effect of an ester versus amide bond on the conformation of a derived macrocycle.
HO2C 2
4
96
6
OR
HO2C 2
R = TBS or TBDPS
4
6
97
NHR
R = Boc
From a retrosynthetic point of view, one has to address the two chiral centers at C-2 and C-6
and the double bond configuration. As both acids contain the same core structure and the
amino acid 97 might be fashioned from the hydroxy acid 96, a divergent synthesis could be
possible. The cleavage of the C4-C5 olefin bond results in two allylic fragments 137 and 138.
The union of these two moieties may be envisaged via a metathesis coupling as shown in
Scheme 37. As previous experience in our lab showed the cross metathesis leads to side
products and does not really work for this alkene combination.[95, 96] An alternative would be
Results and Discussion
69
alkylation of propionyl oxazolidinone 100 with alkyl iodide 139. However, in this strategy
synthesis of the alkyl iodide would require too many steps and costly the Roche ester as a
starting material. Therefore, it was decided to use the Ireland-Claisen rearrangement as a key
step to synthesize the hydroxy acid 96, as it has been reported in previous syntheses of
jasplakinolide hydroxy acid 3. This way the synthesis of hydroxy acid 96 should require only
5 steps. Evans syn aldol reaction[97] between N-propionyl oxazolidinone ent-100 and
methacrolein 141 would provide the chiral center at C-6. The ester 140 could be obtained from
aldol product 142 by subsequent reduction, monoprotection and acylation. Finally, the IrelandClaisen rearrangement of ester 140 would provide the chiral centre at C-2 and as well as the Econfiguration around the double bond.
metathesis
HO2C
OR
137
O
O
alkylation
O
HO2C
138
I
N
OR
OR
Bn
100
96
Ireland-Claisen
rearrangement
139
O
O
O
O
H O
OR
140 R = TBS or TBDPS
141
O
N
Bn
ent-100
Scheme 37: Possible retrosynthetic cuts for hydroxy acid 96.
4.2.5 Synthetic pathway for hydroxy acid 96
The synthesis of the hydroxy acid 96 was started with a known syn selective aldol
reaction between commercially available methacrolein 141 and propionyl oxazolidinone ent100.[97] The propionyl oxazolidinone ent-100 was synthesized from S-phenylalanine using the
same procedure as shown in Scheme 29. The asymmetric Evans aldol reaction of methacrolein
70
Results and Discussion
141 with N-propionyl oxazolidinone ent-100 in the presence of dibutylboron triflate as Lewis
acid and triethylamine as base at -78 oC produced the syn aldol product 142 in 75% yield
(Scheme 38). A subsequent reductive removal of the chiral auxiliary using sodium
borohydride (2.3 M in H2O) in THF (0.1 M) at 0 oC afforded the 1,3-diol 143 in 86% yield.
Monoprotection of the primary alcoholic group with TBDPS-Cl in presence of imidazole at 23
o
C provided the mono protected silyl ether 144 in 97% yield. Subsequent acylation of alcohol
using n-propionyl chloride in presence of pyridine at 0 oC provided the desired ester 140 to
perform Ireland-Claisen rearrangement in 87% yield.
O
O
O
O
OH O
n-Bu2BOTf, Et3N
H
N
O
CH2Cl2, -78 oC
75%
Bn ent-100
141
NaBH4 in H2O
N
O
142 Bn
THF, 0 oC
85%
O
OH
OH
n-propionyl
chloride
TBDPS-Cl, imidazole
OH
143
DMF, rt
97%
OTBDPS
144
pyridine, CH2Cl2,
0 oC, 87%
O
OR
140 R = TBDPS
Scheme 38: Synthesis of ester 140.
Ireland-Claisen rearrangement of the ester 140 would provide the required chiral center at C-2.
The configuration at C-2 will depend on the geometry of enolate. The speciality of the IrelandClaisen rearrangement is that the geometry of the enolate can be controlled by using the
appropriate reaction conditions. Thus, one can obtain selectively both configurations at the
new chiral center by changing the solvents. In our case, Z-enolate will provide the required
configuration at C-2 through a six-membered chair like transition state (Figure 4.2.2). As
reported in the literature,[63] LDA in THF/HMPA was used as base to generate the Z-enolate.
Results and Discussion
71
H
H
heating
O
R
H3C
144
OSiMe3
HO2C
O
Me3Si
R
R
H3C
O
145
146
Figure 4.2.2: Possible transition state to from the required configuration from Z-enolate.
As we discussed earlier in section 2.5.4, the solution of ester 140 in THF (1.0 M) was added to
a solution of LDA in THF/HMPA (7:3) at -78 oC. After 10 minutes, a solution of TBDMS-Cl
in THF (nearly 2.0 M) was added to the reaction mixture at -78 oC in order to trap the enolate
as silylketene actal. The rearrangement followed smoothly and formed the required acid in
75% yield but with very poor diastereomeric ratio (3:1) at C-2 and it was almost impossible to
separate the two diastreomers by column chromatography. The formation of Z-enolate
depends on some important factors such as the solvent and the reaction time as the formation
of the Z-enolate is a thermodynamically controlled reaction. Therefore, we decided to stir the
reaction mixture longer prior to the addition of TBDMS-Cl. This time, the solution of
TBDMS-Cl was added after 40 minutes and this logic worked nicely and high
diasteroselectivity was observed with more than 95% of the desired product with 72% yield
(Scheme 39). The 1H NMR (Figure 4.2.3) of both the diastereomeric mixture and the pure
compound 96 illustrate the obtained results.
72
Results and Discussion
1. LDA, THF/HMPA 7:3
-78 oC, 10 min
2. TBDMS-Cl, -78 oC to rt
then heated to reflux
75%
O
O
HO2C
OR
96
3:1 diastereomeric mixture at C-2
OR
1. LDA, THF/HMPA 7:3
-78 oC, 40 min
R = TBDPS
HO2C
2. TBDMS-Cl, -78 oC to rt
then heated to reflux
72%
OR
96
more than 95% de
Scheme 39: Ireland-Claisen rearrangement of ester 140 under different reaction conditions.
The highlighted regions of the spectra (Figure 4.2.3) correspond to the methyl protns of the
hydroxy acid 96 at C-2 (0.95 ppm), C-6 (1.09 ppm) and C-4 (1.56 ppm) positons, respectively.
3.07
1.60
1.55
ppm
1.02
4.11
1.50
1.10
1.09
1.08
ppm
1.07
3.04
0.95
ppm
1
H NMR spectrum of hydroxy
acid 96 with 3:1 diastereomer
8.0
7.5
7.0
6.5
6.0
5.5
5.0
4.5
4.0
ppm
3.5
3.0
2.5
2.0
1.5
1.0
Results and Discussion
73
1
H NMR spectrum of pure hydroxy
acid 96
3.01
0.05
2.96
1.60
0.95
ppm
1.55
ppm
3.02
1.15
1.10
ppm
8.0
7.5
7.0
6.5
6.0
5.5
5.0
4.5
4.0
ppm
3.5
3.0
2.5
2.0
1.5
1.0
0.5
Figure 4.2.3: 1H NMR spectra of hydroxy acid 96 obtained under two reaction conditions.
4.2.6 Retrosynthetic analysis of amino acid 97
With a good route to hydroxy acid 96 available, we thought it might be the simplest to
substitute the hydroxyl function with an amine equivalent (for example azide). However, this
strategy would require several protection and deprotection steps. As an alternative, we
considered introducing the amino function earlier, for example on primary alcohol 150
(Scheme 40). Ireland-Claisen rearrangement of the ester 147 would provide the amino acid 97.
The ester 147 could be prepared from the protected amino alcohol 148 by deprotection of the
alcohol and acylation with n-propionyl chloride. The protected amino alcohol 148 might be
obtained by Staudinger reaction of azide 149 followed by protection of the resulting amine. In
turn, the azide 149 could be traced back to alcohol 150 and the aldol product 142.
74
Results and Discussion
O
HO2C
NHR
OTBS
O
Ireland-Claisen
rearrangement
NHR
1. deprotection
NHR
2. acylation
1.Staudinger
reaction
148
147
97
1.tosylation
or mesylation
OTBS
N3
2. protection
149
O
OH O
OTBS
1. protection
OH
2. azidation
R = Boc
N
O
2. reduction
150
142 Bn
Scheme 40: Retrosynthetic analysis of amino acid 97.
4.2.7 Synthetic pathway for amino acid 97
The synthesis of amino acid 97 started with the syn aldol product 142. Silylation (1.6
equiv TBSOTf, 2.5 equiv of 2,6-lutidine at 23 oC, 12 h) of the secondary alcohol led to the
silyl ether 151 in 90% yield. Reductive removal of the chiral auxiliary using sodium
borohydride in THF at 0 oC afforded the secondary alcohol protected 1,3-diol 150 in 86%
yield. Tosylation of 150 using tosyl chloride in presence of pyridine at 0 0C afforded the
primary tosylate 152 in 92% yield. Attempts for the synthesis of azide 149 using sodium
azide[98] at 80 oC in DMSO led to decomposition of the starting material with a very bad smell.
The same was observed with mesylate 153. Using DPPA/DBU conditions to synthesize the
azide 149 led to the formation of the corresponding phosphoryl ester along with some side
products. The reason for the failure of the reaction might be due to the formation of [1,3]dipolar cycloadduct.
Results and Discussion
O
OH O
N
75
TBSOTf, 2,6-Lutidine
O
Bn
R = TBS
Bn
142
151
OTBS
NaN3, DMSO
OR
MsCl, Et3N
CH2Cl2, 0 0C
OTBS
NaBH4 in H2O
N
CH2Cl2, rt, 90%
TsCl, pyridine
93%
or
O
OR O
O
OH
THF, 0 oC
86%
150
OTBS
N3
80 0C
149
152 R = Ts
153 R = Ms
Scheme 41: Attempts for the synthesis of azide 149.
At this point, it became necessary to consider other synthetic possibilities for the synthesis of
amino acid 97 avoiding the azide pathway. The synthesis of amino acid 97 requires mainly
introduction of an amine group, because the remainder of the synthesis will build upon the
aldol reaction and Ireland-Claisen rearrangement. The amine could be synthesized by
reduction of an amide which in turn should be available from the corresponding acid.[99]
Keeping this strategy in mind, a second retrosynthetic plan was proposed for amino acid 97
with the amide 154 as key intermediate (Scheme 42). The protected amino alcohol 148 would
be synthesized from the amide 154 by reduction and protection of the resulting amine. The
amide 154 would be prepared from the carboxylic acid 155 via amidation using a suitable
ammonia source.[99]
OTBS
NHR
148
OR O
1. reduction
amidation
OR O
NH2
2. protection
R = Boc
154
OH
155
O
OH O
1. protection
N
2. oxidative
cleavage
O
142 Bn
Scheme 42: Retrosynthetic analysis for amino acid 100 through amide 154.
76
Results and Discussion
In this way, the synthesis of amino acid 97 started from the silylated syn aldol product 151 as
shown in Scheme 44. Oxidative cleavage of 151 using H2O2/LiOH conditions provided the
carboxylic acid 155 in 80% yield. The carboxylic acid 155 was converted to amide 154 using
EEDQ 156 and (NH4)HCO3 at room temperature in 72% yield.[100,
101]
The same
transformation can be accomplished with ethyl chloroformate and triethylamine followed by
NH3 or one can use coupling reagents like EDC (N-Ethyl-N'-(3-dimethylaminopropyl)carbodiimide hydrochloride) followed by NH3.[99] But, in this case the reaction did not work
with the EDC/NH3 conditions.
R
O
O
OC2H5
N
C2H5
O
N
C2H5
O
O
O
O
O
R
R
O
O
O
C2H5
N
O
NH3.H2CO3
O
R
N
NH2
C2H5O
OC2H5
O
EEDQ 156
Scheme 43: Possible mechanism for the formation of an amide from acid using EEDQ 156.
Reduction of amide 154 using LiAlH4 (1.0 M solution in ether) under reflux for 1 h produced
the desired amine 157 but with concomitant deprotection of the alcohol as shown in Scheme
44. As the alcoholic functional group will not affect the protection of the amine using Boc
anhydride, the crude amino alcohol was protected by using Boc-anhydride in presence of
triethylamine providing the Boc protected amino alcohol 158 in 55% yield over two steps.
Acylation of 158 using n-propionyl chloride in the presence of pyridine provided the required
ester 147 for Ireland-Claisen rearrangement in 90% yield. LDA in THF/HMPA (7:3) was
employed for the Ireland-Claisen rearrangement of the ester 147. But the reaction did not work
and the starting material was recovered. The reason might be that the deprotonation occurred
on the amine since only 1.1 eq of base was used for this rearrangement. Thus, the
deprotonation of ester 145 requires more than two equivalents of base. Infact, Ireland-Claisen
rearrangement using NaHDMS (6.0 eq) in THF/HMPA (7:3) at -78 oC, trapping the enolate
Results and Discussion
77
with TMS-Cl and acidic work-up produced the desired amino acid 97 in 58% yield with
excellent diastereoselectivity (> 96%).
O
OR O
OR O
EEDQ, NH4HCO3
30 wt%H2O2, LiOH.H2O
N
O
151 Bn
R = TBS
OH
THF/H2O, 0 oC
80%
155
LiAlH4 (1M in ether)
THF, reflux
OR O
OH
n-propionyl
chloride
NHR
NH2
154
CHCl3, rt
75%
(Boc)2O, Et3N
CH2Cl2, rt
55% (2 steps)
pyridine, CH2Cl2,
0 oC, 87%
157 R = H
158 R = Boc
O
1. NaHDMS, THF/HMPA
-78 oC, 40 min
O
NHR
147
HO2C
NHBoc
2. TMS-Cl, NEt3
-78 oC to rt, then
at 50 oC, 58%
97
Scheme 44: Synthetic scheme for amino acid 97.
4.2.8 Synthesis of jasplakinolide (geodiamolide) analogues using 96 and 97
As we discussed earlier in Section 2.3, the comparative modeling studies of doliculide,
jasplakinolide, and chondramide C revealed that the isopropyl group occupies the same
volume as the phenyl group and the indole ring occupies the same volume as the benzyl group
in space. On the basis of these considerations, in the design of analogues 159 and 160 of
jasplakinolide we decided to replace the unusual amino acids N-methyl-2-bromo D-tryptophan
and β-D-tyrosine with N-methyl-D-tyrosine and L-valine respectively and to substitute the
polypropionate portion with the novel hydroxy and amino acids 96 and 97, respectively to
form depsipeptide 159 and macrolactam 160. The conformational studies of these macrocycles
78
Results and Discussion
might provide some information regarding the influence of amide and ester bond on the
solution structures. This was the idea behind the synthesis of the novel hydroxy- and amino
acids 96 and 97.
O
HN
HO
N
O
O
HN
O
O
H
N
HO
N
H
N
O
O
O
O
N
H
160
159
Figure 4.2.4: Analogues of jasplakinolide (geodiamolide) containing hydroxy- and amino
acids 96 and 97.
As both analogues contain the same tripeptide, it was thought to synthesize the tripeptide
fragment and couple it with the corresponding acids. Initially it was planned to synthesize
analogue 159 using a classical Boc strategy, which would require a macrolactonization in the
final steps. As the macrolactonization failed to give good yields in the total syntheses of
jasplakinolide and geodiamolide, it was planned to combine a dipeptide fragment with an
amino acid (valine)-hydroxy acid fragment which would allow for a more facile
macrolactamization (Scheme 45).
Results and Discussion
79
A
A
O
HN
O
O
D
HO
N
O
N
TBSO
B
O
N
C H
TBSO
N
N
C H
O
B
Boc
O O
tBu
161
OH
O
O
O
O
D
N
C H
159
D
O
HN
A
O
H2N
A
O
Boc
OH
O
tBuO
A+B 163
N
O
Boc
HO
HO
O
O
D
TBSO
C+D 162
OH
FmocHN
tBuO
O
N
C H
B
Boc
Scheme 45: Coupling strategy for the synthesis of the analogue 159.
The N-methyl D-tyrosine derivative D was synthesized from D-tyrosine methyl ester 164 in
three steps.[102] First step was the protection of amine group using Boc anhydride, followed by
silylation of the hydroxy function as silyl ether yielding fully protected tyrosine 166 as shown
in Scheme 46. N-methylation of 166 by using NaH (60% dispersion in mineral oil) for
deprotonation and MeI conditions at 0 oC afforded the N-methyl D-tyrosine derivative 167 in
55% yield over 3 steps. Deprotection of the Boc group of 167 with trifluoroacetic acid (TFA)
provided the corresponding free amine. PyBroP mediated coupling of the crude amine with NBoc-L-alanine provided the dipeptide 168 in 55% yield.[81, 103] The subsequent hydrolysis of
the methyl ester on dipeptide 168 posed a challenge since conditions would have to be found
that leave the phenolic silyl ether intact. Basic hydrolysis of dipeptide 168 using NaOH (0.4
N) led to hydrolysis as well as cleavage of the silyl group on the phenol. Therefore, the mild
reagent trimethyl tin hydroxide was used for the hydrolysis.[104, 105] Indeed, the methyl ester
cleaved without disturbing the silyl group on the phenol giving the desired acid 162.
80
Results and Discussion
OMe
TBS-Cl, imidazole
DMAP (cat)
OMe
(Boc)2O, NEt3
HO
O
NH2.HCl
HO
164
OMe
55% (3 steps)
166
OMe
TBSO
168
TBSO
N
167
Boc
OH
Me3SnOH, 1,2-DCE
O
O
N
H
1. TFA, CH2Cl2, 0 oC
O
OMe
N
DMF
165
NaH (60% dispersion
in oil), MeI, THF, 0 oC
O
NHBoc
TBSO
O
NHBoc
dioxane, H2O
o
80 C, 1 h
Boc
N
TBSO
162
2. N-Boc-L-Ala, PyBroP,
DIEA, CH2Cl2,
0 oC, 55%
O
O
N
H
Boc
Scheme 46: Synthesis of dipeptide fragment 162.
The other fragment A+B 163 could be obtained from the hydroxy acid 96 and Fmoc-L-valine
in four steps. As the linear depsipeptide needs the deprotection of both the amine and the
carboxylic group prior to cyclization, it would be ideal having similar protecting groups on
both sides, which could be cleaved in a single step. As the dipeptide 162 has a Boc-protecting
group at the N-terminus, it would be ideal to have a tert-butyl group on the C-terminus, so that
both groups could simultaneously be cleaved by TFA. The synthesis of fragment 163 was
started with the protection of hydroxy acid 96 with a tert-butyl group as shown in Scheme 47.
DCC mediated esterification of hydroxy acid 96 with tert-butanol in presence of DMAP as
catalyst provided the desired product 169 in very low yield around 25%. Addition of higher
amounts of the catalyst did not improve the yield. Then it was decided to employ the
Yamaguchi esterification using 2,4,6-trichlorobenzoyl chloride and DMAP. Esterification of
hydroxy acid 96 with tert-butanol using Yamaguchi conditions with excess of reagents
provided the desired protected acid 169 in almost quantitative yield.[70] After obtaining the
protected hydroxy acid 169, the TBDPS group was removed in order to couple the resulting
alcohol with Fmoc-L-valine. Deprotection of the silyl group using TBAF provided the
hydroxy ester 170 in 90% yield. DCC mediated esterification of Fmoc-L-valine with hydroxy
compound 170 furnished the ester 171 in 88% yield. Finally, removal of the Fmoc group using
Results and Discussion
81
diethylamine led to the desired amine 163 in 88% yield. This way the fragment A+B was
obtained.
HO2C
2,4,6-trichlorobenzoyl
chloride
,
tBuO2C
Et3N, DMAP, tBuOH
OR
toluene, -78 C to rt
97%
96 R = TBDPS
Fmoc-L-Valine,
DCC, DMAP
tBuO2C
OH
THF, 0 oC
88%
169 R = TBDPS
O
tBuO2C
CH2Cl2, 0 C,
88%
NHFmoc
O
o
170
TBAF
OR
o
171
O
Et2NH, THF
tBuO2C
O
NH2
0 oC, 88%
163
Scheme 47: Synthesis of amine fragment 163.
TBTU mediated coupling of the fragments 162 and 163 provided the desired linear depsipetide
161 in 55% yield, but as a diastereomeric mixture with 3:2 ratio (Scheme 48). Careful
inspection of the 1H- and
13
C NMR spectra of 161 showed that several peaks were doubled.
Even TLC showed two clear spots of the diastereomeric mixture. Hence, rotamers around the
boc-group could be ruled out. This result was very unusual as the amine 163 was
diastereomerically pure and TBTU mediated coupling in our experience always provided the
coupled product without racemization.
OH
TBSO
O
O
N
N
C H
162
O
H2N
Boc
O
HN
O
TBTU, HOBt,
O
DIEA, DMF, rt
55%
TBSO
tBuO
163
Scheme 48: Synthesis of linear depsipeptide 161.
N
O
O
N
H
O
Boc
161
3:2 diasteromers
O
OtBu
82
Results and Discussion
We suspected that the problem might lie within the dipeptide acid part 162. It was thought that
racemization might have occured in the hydrolysis step (Scheme 46). In order to probe this
hypothesis, the methyl ester was replaced with the benzyl ester which should allow for a
racemization free hydrolysis. As the dipeptide acid 162 was known from literature,[102] we
decided to follow the same strategy for the synthesis of dipeptide acid 162 as in the literature.
For this purpose, D-tyrosine benzyl ester 172 was chosen and were followed similar steps as in
Scheme 46 to the fully protected D-tyrosine derivative 173. Subsequent N-methylation of 173
using NaH (60% dispersion in mineral oil) followed by addition of MeI provided the Nmethyl–D-tyrosine derivative 174. Deprotection of the Boc group using TFA and PyBroP
mediated coupling of the resulting amine with N-Boc-L-alanine provided the dipeptide 175.
Subsequent hydrogenation led to the dipeptide acid 162. TBTU mediated coupling of the acid
162 with amine 163 provided the same result as in Scheme 48. The optical rotation of the
dipeptide 175 was measured in order to compare the optical purity with the known literature
value; surprisingly both values had no comparison. So the total problem lied somewhere in the
synthesis of dipeptide and not the coupling step to 161. It might be possible that the
racemization had occured during the N-methylation of the D-tyrosine derivative. To prove
this, a series of experiments were performed (Scheme 49 and 50). The above sequence was
repeated with L-tyrosine benzyl ester ent-172 in order to synthesize the dipeptide consisting of
L-tyrosine and N-Boc-L-alanine. The synthesis was done in order to compare the NMR
spectra of both compounds 175 and 176.
Results and Discussion
1. (Boc)2O, NEt3,
dioxane, H2O
OBn
O
NH2.HCl
HO
172 D-tyrosine
ent-172 L-tyrosine
OBn
O
N
TBSO
Boc
174
ent-174
83
NaH (60% dispersion
in oil), MeI, THF, 0 oC
OBn
2.TBS-Cl, Imidazole
DMAP (cat), DMF
84%
O
NHBoc
TBSO
55% (3 steps)
173
ent-173
OBn
1. TFA, CH2Cl2, 0 oC
2. N-Boc-L-Ala, PyBroP,
N
TBSO
DIEA, CH2Cl2,
0 oC, 55%
175 D-Tyr-L-Ala
176 L-Tyr-L-Ala
O
O
N
H
Boc
OH
175
H2, Pd/C, EtOH
rt, 1 h, 99%
N
TBSO
162
O
O
N
H
Boc
Scheme 49: Synthesis of dipeptide acid 162 from tyrosine benzylester 172.
Finally, the L- and D- tyrosine derivatives 174 and ent-174 were prepared by a differenet
method (Scheme 50).[106] The idea here was to see whether the N-methylation step caused
racemization. In this pathway the syntheses of dipeptides 175 and 176 were started with the
commercially available N-Boc-D- and L-tyrosine 177 and ent-177. Protection of the phenol
group using TBDMS-Cl in presence of imidazole produced the disilylated products 178 and
ent-178. Treatment of silyl esters with 1 N K2CO3 cleaved the silyl group on the acid leading
to the phenol protected products 179 and ent-179. N-Methylation of 179 and ent-179 was
achieved by producing the dianion using 2.5 eq of t-BuLi. Treatment of 179 and ent-179 with
t-BuLi at -78 oC and subsequent addition of MeI produced the N-methyl tyrosine 180 and ent180 derivatives in good yields. Subsequent protection of the acid function with benzyl alcohol
using DCC in presence of DMAP as catalyst gave the esters 174 and ent-174. Subsequent
removal of the Boc group using TFA in CH2Cl2 at 0 oC gave the crude amine, and PyBroP
mediated coupling of the crude amine with N-Boc-L-alanine in presence of Hünig’s base
furnished the corresponding dipeptides 175 and 176, respectively. Surprisingly, the products
174 and ent-174 that were synthesized according to Scheme 50 have nearly the same optical
84
Results and Discussion
rotation values with different signs +71.8 and -69.2. In contrast, optical rotation value of the
compounds 174 and ent-174 (both Land D) synthesized via Scheme 49 showed zero. But, the
optical rotation of the tyrosine derivative 173 (before N-methylation step) correlated with the
literature value.[102] So, at this point we can clearly say that the base NaH (60% dispersion in
mineral oil) caused the racemization at the α-chiral center in both the benzyl and the methyl
esters. The 1H NMR spectra of dipeptides 175 and 176 derived from both Schemes 49 and 50
clearly show the racemization pattern. The expanded regions of the spectra clearly showed the
different chemical shift values for the chiral methyl groups (0.85 ppm in 175 and 1.18 ppm in
176) in the compounds 175 and 176 (Figure 4.2.5).
OTBS
TBS-Cl, Imidazole
DMAP (cat)
OH
O
NHBoc
DMF, rt
TBSO
HO
177
D-tyrosine
ent-177 L-tyrosine
O
H
N
179
ent-179
rt, 86% (2 steps)
178
ent-178
OH
TBSO
1N K2CO3, THF
O
NHBoc
Boc
OBn
OH
t-BuLi, MeI, THF
O
o
-78 C to rt,
76%
TBSO
Me
N
Boc
180
ent-180
BnOH, DCC, DMAP
CH2Cl2, 0oC
89%
OBn
o
1. TFA, CH2Cl2, 0 C
O
N
TBSO
174
ent-174
Boc
2. N-Boc-L-Ala, PyBroP,
DIEA, CH2Cl2,
0 oC, 55%
N
TBSO
O
O
175
176
N
H
Boc
Scheme 50: Synthesis of dipeptides 175 and 176 using D and L-tyrosine derivatives 177 and
ent-177.
Results and Discussion
85
Figure 4.2.5 a) 1H NMR
spectrum of the compounds
175 and 176 synthesized by
Scheme 49
N-methyl
6.00
2.80
ppm
2.75
2.99
0.90
0.85
0.80
ppm
0.75
0.70
2.87
1.20
7
1.15
ppm
6
5
4
3
2
1
0
ppm
N-methyl
b) 1H NMR of 175 synthesized
by Scheme 50
2.74
2.80
ppm
2.75
3.00
0.90
7
6
0.85
0.80
ppm
0.75
5
4
3
2
1
0
ppm
N-methyl
3.02
c) 1H NMR spectrum of 176
synthesized by Scheme 50
2.80
ppm
3.03
1.20
1.15
ppm
7
6
5
4
3
ppm
2
1
0
86
Results and Discussion
After proving that the racemization occurred in the N-methylation step with the base NaH
(60% dispersion in mineral oil), the dipeptide 175 was then synthesized according to the
Scheme 50. The dipeptide 175 was subjected to hydrogenation using Pd/C as catalyst in order
to remove the benzyl protecting group resulting in the diastereomerically pure free acid
compound 162 (Scheme 51). TBTU mediated coupling of 162 with amine 163 provided the
linear depsipeptide 161 in 55% yield. Both protecting groups could now be removed in one
step by using trifluoroacetic acid. After concentration of reaction mixture, the macrolactam
formation was carried out in DMF (0.001 M) using TBTU in presence of HOBt at room
temperature which led to the formation of the TBS protected cyclic depsipeptide. A final
deprotection using TBAF gave the desired cyclic depsipeptide 159.
OH
OBn
O
O
N
TBSO
H2, Pd/C
Boc
N
H
175
N
TBSO
O
O
163, TBTU, HOBt,
Boc
N
H
162
O
HN
TBSO
EtOH, rt
99%
N
O
O
N
H
1. TFA, CH2Cl2, 0 C
Boc
2. TBTU, HOBt, DIEA,
DMF, rt, 40%
3. TBAF, THF, 0 oC
80%
O
OtBu
O
HN
o
O
DIEA, DMF, rt
51%
HO
161
N
O
O
O
O
N
H
159
Scheme 51: Synthesis of analogue 159.
In order to reach the amide analogue 160, it was decided to assemble tripeptide 182 and to
combine it with amino acid 97 to get the seco-compound 181, which on macrolactamization
should provide macrolactam 160 (Scheme 52).
Results and Discussion
A H
N
HN
D
HO
N
87
O
O
HN
O
O
B
TBSO
CO2Me
NHBoc
O
O
N
NH
N
C H
160
O
181
NHBoc
OH
HN
TBSO
HO2C
97
N
CO2Me
O
O
N
182 H
TBSO
Boc
N
O
O
N
H
OMe
H2N
O
Boc
L-valine methylester
162
Scheme 52: Coupling strategy for the analogue 160.
The synthesis of analogue 160 was started with the dipeptide acid 162 which was synthesized
according to Scheme 50. TBTU mediated coupling of 162 with L-valine methylester
hydrochloride salt in presence of HOBt produced the tripeptide 181 in 71% yield. At this
point, the Boc protecting group at the N-terminus was removed with trifluoroacetic acid,
followed by amide formation of the resulting amine with amino acid 100 using TBTU to
produce the linear tetrapeptide 181. Ester hydrolysis of 181 not only cleaved the ester function
but also the silyl phenyl ether. Boc deprotection of the resulting product using TFA produced
the seco-acid which on macrolactamization with TBTU in presence of HOBt produced the
desired macrolactam 160 (Scheme 53). The detailed conformational analysis of the
compounds 159 and 160 will be discussed in the conformational analysis section.
88
Results and Discussion
OH
TBSO
O
O
N
N
H
HN
L-val methylester,
TBTU, HOBt, DIEA
DMF, rt, 71%
TBSO
Boc
Boc
2. 97,TBTU, HOBt,
DIEA, DMF, rt, 53%
CO2Me
HN
N
1. TFA, CH2Cl2, 0 oC
O
O
N
H
182
162
TBSO
N
CO2Me
NHBoc 1. LiOH, THF, H O, rt
2
O
O
2. TFA, CH2Cl2, 0 oC
NH
O
H
N
HN
HO
N
3. TBTU, HOBt, DIEA,
DMF, rt, 45%
181
O
O
O
O
N
H
160
Scheme 53: Synthesis of jasplakinolide analogue 160.
4.2.9 Synthesis of Nor-methyl Chondramide C
The chondramides are cyclodepsipeptides produced by strains of myxobacterium,
Chondramyces crocatus.[107,
108]
The structures of the chondramides published so far are
related to jasplakinolide. Besides different substituents, the chondramides have an 18membered macrocyclic ring instead of the 19-membered ring system of jasplakinolide. Both
compounds inhibit the growth of yeasts and show cytostatic activities. Chondramide C, which
is quite similar to jasplakinolide, appears to have antiproliferative activity against carcinoma
cell lines by targeting the actin cytoskeleton.[20, 109] Structurally and activity wise chondramide
C is similar to jasplakinolide. Chondramide C contains a tripeptide similar to jasplakinolide
except for the bromine on the indole ring and a hydroxy acid unit which is one carbon shorter
than the jasplakinolide hydroxy acid. The hydroxy acid of chondramide C also has non bonded
interactions like the jasplakinolide hydroxy acid. As the configuration of chondramide C was
not yet fully determined, but based on the similar biological activity and same binding site as
the jasplakinolide, the configuration of chondramide C proposed was similar to the one of
jasplakinolide.[110]
Results and Discussion
89
OH
OTBS
R = Me or tBu
O
O
H
H
N
Me
N
H
N
O
O
O
N
H
N
Me
H
O
chondramide C (5)
N
OR
N
HO
O
O
H
N
Boc
184
HO
O
183
18-membered
Scheme 54: Retrosynthetic analysis of the proposed structure of chondramide C.
The hydroxy acid 183 of chondramide C has four methyl groups and one double bond in
which three of the methyl groups are placed in 1,3-distance to each other giving rise to one syn
pentane interaction and one allylic strain which make the molecule less flexible. Two methyl
groups are placed in 1,2-distance so that the rotation of methyl groups around the single bond
might be restricted. Thus, the hydroxy acid 183 should have a definite conformation. If one
compares the hydroxy acid of chondramide C and hydroxy acid 96, one can recognize that
they have the same chain length and differ only by the methyl group at C-7. Thus, it was
interesting to compare the biological activity of the chondramide C analogue containing the
same tripeptide and hydroxy acid 96 which we call as nor methyl chondramide C 185.
As nor methyl-chondramide C contains the tripeptide and hydroxy acid parts, it was decided to
assemble the tripeptide 184 and to combine it with the ester 170 to form the linear
depsipeptide which on macrolactam formation would provide the desired product 185
(Scheme 56). The tripeptide part contains three different amino acids; β-D-tyrosine derivative,
N-methyl D-tryptophane and L-alanine. As the tripeptide is similar to the tripeptide part in the
analogue 134, it was decided to follow a similar strategy to construct the tripeptide. The
necessary β-D-tyrosine derivative 186 was synthesized according to a known literature
90
Results and Discussion
procedure[111] using an Arndt-Eistert reaction as the key step. The dipeptide 187 was
synthesized according to a known literature procedure as well.[112]
OH
OTBS
R = Me or tBu
O
O
H
H
N
Me
H
N
N
H
O
O
O
N
Me
N
N
H
N
Boc
O
O
H
N
Boc
186
tBuO
O
170
184
normethyl chondramide C 185
OTBS
OMe
N
Me
N
O
O
H
N
Boc
OMe
187
HO
O
O
H
H
OR
N
H
OMe
N
O
Me
N
H
HO
O
H
N
Boc
188
Scheme 55: Coupling strategy for normethyl chondramide C 185.
The synthesis of β-D-tyrosine derivative 186 started with the silyl ether of p-hydroxy N-BocL-phenyl glycine 189 as shown in Scheme 56. Accrording to a recent literature procedure, a
commercially available solution of trimethylsilyl diazomethane in ether was employed to
produce the diazoketone 190 instead of using diazomethane.[113] The acid 189 was treated with
ethyl chloroformate in presence of triethylamine to produce the mixed anhydride. To this
mixed anhydride a solution of trimethylsilyl diazomethane in ether was added in order to
produce the diazoketone 190. However, all attempts failed to produce the diazoketone with
trimethylsilyl diazomethane. There was no other way except using diazomethane to synthesize
the compound 190 from acid 189. Accordingly, a freshly prepared solution of diazomethane
solution in ether was added to the crude reaction mixture consisting the mixed anhydride at 0
o
C. The crude product 190 was almost pure on TLC after work up, so it was used for the Wolf-
Results and Discussion
91
rearrangement without any purification. Rearrangement occurred without any problem with
methanol in presence of silver benzoate, producing the desired product 186 in 80% yield
(Scheme 56). The β-tyrosine derivative 186 was treated with trifluroacetic acid in order to
deliver the crude amine 191.
OTBS
OTBS
OTBS
1. ClCO2Et, NEt3, ether,
0 oC, 1h
H
N
Boc O
OH
MeOH, silver benzoate,
o
-30 C
H
2. CH2N2 in ether
0 oC to rt
N
Boc O
189
O
H
80%
N2
OMe
N
Boc
186
190
OTBS
TFA, CH2Cl2, 0 oC
O
OMe
H2N
191
Mechanism:
H2C N N
O
R
H
N
Boc O
Cl
R
O
H
O
R
H
H
N
Boc O
N
R
H
N
Boc
N
N
Boc O
R
O
O
H
Et
O
R
H
N
Boc O
Ag
N
N
N
Boc O
N
N
R
H
N
Boc
C
O
HOMe
Ketene
OTBS
O
OMe
R=
Scheme 56: Synthesis and mechanism for the formation of β-tyrosine derivative 186.
The synthesis of dipeptide 187 started with N-methyl D-tryptophane methyl ester 188 (Scheme
57). PyBroP mediated coupling of N-Boc-L-alanine with N-methyl D-tryptophane methylester
92
Results and Discussion
188 in presence of diisopropylethylamine produced the desired dipeptide 187. At this point,
the methyl ester at the C terminus was cleaved to produce the dipeptide acid 192. TBTU
mediated coupling of dipeptide acid 192 and β-amino ester 191 in presence of HOBt produced
the desired tripeptide 184 in 70% yield. Trimethyl tinhydroxide[104, 105] mediated hydrolysis of
tripeptide 184 furnished the tripeptide acid which on esterification using DCC in presence of
catalytic amounts of DMAP with hydroxy acid derivative 170 gave the seco compound 193 in
80% yield for two steps. Trifluoroacetic acid mediated cleavage of both protecting groups Boc
and tBu and subsequent macrolactam formation using TBTU in presence of HOBt in DMF at
room temperature led to the TBS protected nor methyl chondramide C which on treatment
with TBAF produced normethyl chondramide C 185 (Scheme 57).
H
OMe
H
N
N-Boc-L-Ala, PyBrop, DIEA
N
N
CH2Cl2, 0 oC, 60%
O
Me
OR
Me
N
H
188
NaOH,
THF/H2O
OTBS
191, TBTU, HOBt,
DIEA, DMF, rt,
70%
O
O
187 R = Me
H
N
Boc
OTBS
192 R = H
O
H
H
N
Me
N
O
OMe
N
O
O
1. Me3SnOH, 1,2-DCE
80 oC
2. 170, DCC, DMAP,
H
CH2Cl2, 0 oC to rt
80%
N
Boc
H
N
Me
N
N
O
O
O
H
N
Boc
O
193
184
1. TFA, CH2Cl2, 0 oC
H
normethyl chondramide C 185
2. TBTU, HOBt, DIEA,
DMF, rt, 44%
3. TBAF, THF, 0 oC
73%
Scheme 57: Total synthesis of normethyl chondramide C 185.
OtBu
Results and Discussion
93
4.2.10 Design and the synthesis of analogues of hydroxy acid 96
Cyclic peptides provide ideal scaffolds for exploring structure-activity relationships in
ligand-receptor interactions. Cyclization serves to increase membrane permeability by
elimination the charged termini and facilitating internal hydrogen bonded conformers.
Cyclization of peptides introduces an extra element of constraint (e.g. double bond, aromatic
ring etc.), which confers conformational restriction to the peptide leading to an entropically
advantageous mode of binding to the target protein.[114] As the hydroxy- or amino acid 96 or
97 contain the central allylic system and a methyl group at the allylic position to the double
bond, the resulting 1,3-allylic interaction makes the molecule less flexible. As we mentioned
in the design of amino acid 95 (Figure 4.2.1), the m-xylol ring can provide a similar restriction
as an allylic system. With this in mind the amino- and hydroxy acids 194 and 195 were
designed in order to construct some cyclic peptides. Recently, some working groups[115-120]
synthesized some small cyclic peptides (Figure 2.4.6) containing an aromatic ring as central
part to mimic β-turns and they successfully created β-turn mimics .
O
R1
HN
O2 N
H
H
N
HN
R2
O
N
O HN
HN
CONH2
O
Ph
O
O
N
H
O
O
NH
O
O
HN
S
Figure 4.2.6: Some examples of peptidomimetics containing aromatic ring as central part.
94
Results and Discussion
O
HO
HO2C
HO2C
OR
NHR
194
OR
R = Boc
O
HO
96
OR
ring closure
195
R = TBS
Scheme 58: Replacement of the allylic system of hydroxy acid 96 with a meta-substituted
aromatic portion.
4.2.11 Retrosynthetic analysis and synthesis of amino acid 194
As one can see amino acid 194 contains a α-methyl carboxylic acid on one side and a
methylamine group on the other side of the benzene ring. It should be possible to synthesize
the α-methyl carboxylic acid by hydrolysis of the Evans alkylation product between propionyl
oxazolidinone and a benzyl bromide derivative. The methylamine group could be obtained by
reduction of a cyano group either by hydrogenation or by hydride reduction. In turn, the cyano
group might be obtained by ipso substitution of the bromide with copper(I) cyanide.[120] Thus,
a suitable electrophile for the synthesis of amino acid 194 might be 3-bromobenzyl bromide.
O
HO
NHR
R = Boc
1. hydrogenation
2. protection
O
100 Bn
CN
N
Bn
O
N
O
O
3. hydrolysis
194
O
O
2. cyanation
196
Br
Br
1. alkylation
197
Scheme 59: Retrosynthetic analysis for amino acid 194.
Results and Discussion
95
The synthesis was started with commercially available 3-bromobenzylbromide 197 and
propionyl
oxazolidinone.
Alkylation
of
propionyloxazolidinone
100
with
3-
bromobenzylbromide using a 2 M solution of NaHDMS at -78 oC produced the corresponding
alkylated product 198 in 50% yield, contaminated with propionyl oxazolidinone 100 in about
10%. The cyanation (SNAr reaction) of the alkylation product 198 using copper(I) cyanide in
DMF at 100 oC produced the corresponding cyanide 196 in 58% yield.[120] Hydrogenation of
the cyanide 196 in presence of Pd/C and formic acid did not provide the corresponding amine
.[119] Attempts to prepare the amine using hydrogenation in different solvents such as ethanol
and ethyl acetate did not provide a good result. It might be possible that due to the steric
crowding of the chiral auxiliary, cyanide can not sit on the surface of the catalyst, thus there is
no hydrogenation. Therefore, the chiral auxiliary was cleaved by in situ generation of methoxy
magnesium bromide using methylmagnesium bromide in methanol to get the corresponding
methyl ester 199 in 55% yield. Hydrogenation of the nitrile 199 in presence of 10% Pd/C and
formic acid in methanol produced the corresponding amine salt in 14 hours at room
temperature. Protection of the amine using Boc-anhydride in presence of 1N NaOH produced
the Boc-amino acid ester 200 in 70% yield. However, this hydrogenation was not really
reproducable. In some runs we observed formation of the over reduced toluene derivative 201.
This result was confirmed by NMR spectra. Even though the synthesis is short, the yields were
quite low in each step and the hydrogenation is not a reliable procedure.
96
Results and Discussion
O
O
O
N
O
Br
Br
Bn
198
11% with chiral auxiliay
197
O
O
O
CN MeOMgBr, MeOH
N
O
CuCN, DMF, 100 oC
Br
N
O
Bn
100
O
NaHDMS at -78 oC
CN
MeO
2. (Boc)2O,
0.4 N NaOH,
dioxane, rt
0 oC, 52%
Bn
196
1. 10% Pd/C, H2 atm,
HCO2H, 14 h, rt
199
O
O
MeO
CH3
MeO
NHBoc
201
200
Scheme 60: Attempted route for the synthesis of amino acid 194.
Since Scheme 60 led to the undesired side product in the hydrogenation, a method was sought
that would provide the amino acid 194 in less number of steps. In the present Scheme, we
decided to use dibromobenzyl derivative 104 to avoid the reduction of the cyanide group. The
key steps involved in the present strategy were an asymmetric Evans alkylation and SN2
displacement, reduction and hydrolysis.
O
HO
NHR
R = Boc
O
O
1. mono alkylation
O
N
N3
3. hydrolysis
2. azidation
Bn
194
O
O
1. hydrogenation
2. protection
202
O
N
100
Bn
Br
Br
104
Scheme 61: Retrosynthetic analysis for amino acid 194 using a mono alkylation strategy.
Results and Discussion
97
The crucial step in the synthesis using Scheme 61 is to achieve a selective mono alkylation. As
we mentioned in the synthesis of the novel ω-amino acid 95, the dialkylated product can be
synthesized using an excess of chiral reagent. It is obvious that using excess alkylating reagent
should produce the mono alkalated product as major product due to reduced availability of the
enolate. Synthesis of amino acid 194 was started with the commercially available benzyl
bromide derivative 104 as shown in Scheme 62. Alkylation of propionyl oxazolidinone 100
with 3 equivalents of benzyl bromide derivative 104 using LDA at -78 oC produced the mono
alkylated product 203 in 60% yield. The excess benzylbromide derivative was recovered by
washing the reaction mixture with hexane as it dissolves well in hexane. The SN2 displacement
of bromide with azide using sodium azide in ethanol at 60 oC afforded the desired azide 202 in
85% yield. The crude azide 202 was almost pure and it was used without further purification.
The reduction of azide[121] to amine in presence of 10% Pd/C and hydrogen atmosphere
produced the desired amine. Subsequent protection of the amine using Boc-anhydride in
presence of 1 N NaOH delivered the Boc protected amino acid derivative 204 in 70% yield.
The hydrolytic cleavage of the resulting product using 30 wt% H2O2 and lithium hydroxide led
to the the desired amino acid 194 in 80% yield. This way the amino acid was secured in less
number of steps with good yields (Scheme 62).
O
O
N
O
O
LDA. -78 oC, THF
O
O
Br
N
NaN3, EtOH, 60 oC
Br
Br
Bn
Bn
100
203
104
O
O
O
1.10% Pd/C, H2 atm,
EtOH, rt
N3
N
Bn
2. (Boc)2O, NaOH,
dioxane, rt, 70%
202
30 wt% H2O2, LiOH,
THF/H2O, 0 oC
O
O
O
N
Bn
O
HO
NHR
85%
R = Boc
194
Scheme 62: Efficient synthesis of amino acid 194.
NHBoc
204
98
Results and Discussion
4.2.12 Synthesis of hydroxy acid 195
Hydroxy acid 195 also has one methyl group α to the carboxylic group. Therefore, the
synthesis of the hydroxy acid 195 should be possible from the mono alkylated product 203.
Hydrolysis of monoalkylated product 203 would generate the carboxylic acid as well as cause
nucleophilic attack of hydroxide at the benzylic position by replacement of the bromide. This
way unprotected hydroxy acid 205 might be obtained in a single step. The protection of the
hydroxy group as silyl ether and basic hydrolysis of the silyl ester should then provide the
desired hydroxy acid 195 in just a few steps.
O
HO
1. protection
OR
O
N
Bn
OH
HO
2. acidic work up or
1N K2CO3
195 R = TBS
O
O
oxidative
cleavage
O
205
Br
203
Scheme 64: Retrosynthetic analysis for hydroxy acid 195.
The synthesis of hydroxy acid 195 started with the mono alkylation product 203. The
hydrolytic cleavage of the chiral auxiliary indeed produced the desired unprotected hydroxy
acid 205 in 70% yield. The protection of hydroxy acid using TBDMS-Cl in presence of
imidazole led to the TBS protected hydroxy acid silyl ester which on treatment with 1M
K2CO3 in THF produced the hydroxy acid 195 in 70% yield (Scheme 65).
Results and Discussion
O
O
O
99
O
30 wt% H2O2, LiOH,
o
Br THF/H2O, 0 C
HO
N
OH
Bn
205
203
O
HO
OTBS
195
Scheme 65: Synthesis of hydroxy acid 195.
1. TBS-Cl, imidazole,
DMF, rt
2. 1M K2CO3, THF
rt, 70%
100
Results and Discussion
4.3 Conformational studies
4.3.1 Conformational studies of jasplakinolide analogues 129, 134, 135 and 136
Aside from variations in the side chains, the major difference of the four analogs 129,
134, 135 and 136 is the replacement of the polypropionate subunit with a m-xylyl containing
amino acid 95. The 19-membered systems 135 and 136 are structurally closely related to the
natural compound jasplakinolide. Compound 136 possesses a secondary amide in position 16
(Figure 4.3.1). 1H and
13
C resonance assignments via DQFCOSY, ROESY/NOESY and
HMQC spectra were performed in DMSO-d6 and gave a single signal set for 135 and a
doubled signal set for 136 with the trans isomer of the N-methylated amide populated for >
99%.
OMe
OMe
2'
2'
3'
3'
O
H 19
H
2'''
1'''
N 1
4N
20
H
17
N
O
N
O
9'''
H
6'''
14 H
8''' 7'''
Me
N
10 4''
13
11
O
O
19
H
H
2'''
1'''
N 1
4N
20
H
17
N
O
N
O
9'''
Me
14 H
8''' 7'''
Me
N
10 4''
13
11
O
analog 136
analog 135
Figure 4.3.1: Structures for the 19-membered ring analogs 135 and 136 with the numbering
systems used.
One large and one small 3JHH coupling constant (Table 3) for the methylene groups in
positions 2, 6 and 10 of 130 and in positions 6 and 10 for 131 are typical for a well defined
gauche-anti orientation with preference for the major rotamer(s) as distinguished by
characteristic ROE signals. This allowed the assignment of the pro-R and pro-S protons of the
Results and Discussion
101
methylene groups. Due to signal overlay a statement on the methylene protons in position 2 of
136 was not possible.
Table 3. 3JHH coupling constants in Hz of the methylene protons in 129, 134, 135 and 136.
Due to identical chemical shift for H10h/H10t of 129 and H2h/H2t of 136, resp. the coupling
constants could not be determined.
129
2
JH1h,H1t
13.5
3
JH1h,H2
3
2
3
134
135
136
JH2h,H2t
13.6
n.d.
3
JH2h,H1
4.3
n.d.
3.6
3
JH2t,H1
10.4
n.d.
13.1
2
JH6h,H6t
13.0
12.9
~ 7 (from COSY)
3
JH6h,H5
10.6
9.0
JH6t,H5
3.7
4.7
14.0
2
n.d.
12.7
JH1t,H2
3.7
JH6h,H6t
13.1
JH6h,H5
6.1
3
JH6t,H5
4.1
~ 5 (from COSY)
3
2
JH10h,H10t
n.d.
12.6
2
JH10h,H10t
11.0
14.0
3
JH10h,H11
n.d.
11.3 (via H11)
3
JH10h,H11
2.7
3.1
3
JH10t,H11
n.d.
4.0
3
JH10t,H11
13.7
10.3
With ROESY and NOESY measurements characteristic proton-proton interactions could be
determined and transferred into proton-proton distances by integration. They are in very good
agreement for compounds 135 and 136 corresponding to a minor influence of the Nmethylation in position 16 on the overall structure. No intensive cross signals were found
between H α-protons of adjacent amino acids, thus proving trans conformation for all amide
bonds. The methylated amide in 136 leads to an energy barrier which gives rise to a separated
signal set for the cis-amide. The signal set for the cis isomer is populated to less than 1%. The
significant preference for the trans rotamer, to such an extent unusual for a tertiary amide, was
found for the natural compound jasplakinolide as well,[122] thus emphasizing the good analogy
of synthetic analog and natural product.
The dynamical properties of the whole macrocycles 135 and 136 are consistent including the
side chains having the same dynamical behavior as the macrocyclic ring. The newly inserted
m-xylyl unit performs an oscillating movement as ROESY data yield only mean proton-proton
102
Results and Discussion
distances for several possible orientations of the phenyl ring in relation to the macrocyclic
ring. Via temperature measurements, which gave temperature coefficients for the amidic NH
protons in the range of –3.7 to –6.0 ppb K-1, transannular hydrogen bonds could be excluded
but nevertheless, as coupling constants and ROESY data show, the 19-membered analogs 135
and 136 are rigid macrocyclic structures.
Within a MD simulation, calculated structures of 135 and 136 are very well comparable with
each other, the only difference being the orientation of the NH proton respectively the Nmethyl group in position 16 with the proton in 135 pointing into the middle of the macrocyclic
ring and the methyl group in 136 oriented towards the lower side of the ring (Figure 4.3.2).
135
136
Figure 4.3.2: Energy-minimized structures for 135 and 136 after a 100 ps MD Simulation at
300 K. In green torsion with the largest difference for 135 and 136 with the N-methyl group in
136 oriented onto the lower side of the ring and the NH proton in position 16 of 135 pointing
into the macrocyclic ring.
The results presented above are in accordance with NMR structural investigations of
jasplakinolide.[122] Jasplakinolide does not possess any hydrogen bonds nor a higher fraction of
cis-amide, either. The 3J coupling constants yield a more flexible structure for the natural
analog in comparison to the compounds 135 and 136, however the difference is small. The
Results and Discussion
103
orientation of the two aromatic side chains were not determined in detail in the study presented
here. But a possible „tweezer“ structure as stated in literature cannot be supported by NOEs
between side chain protons.
The 18-membered rings of compounds 129 and 134 show several structural differences when
compared to jasplakinolide. They are rather analogs of geodiamolide with a difference in the
position of the aromatic amino acid (Figure 4.3.3).[121]
4'
15
H
I
HO
24
3
Me
15
N
5
N
1
3'
O
H
19 N
1 O
O
O
N
Me
17
21
11
H
7
O
Geodiamolide (2)
20
H
2
O
N
O
16
N
O
14
H
Me 13 N
10
11
O
H
5
6
6''
4''
analog 129
Figure 4.3.3: Structures for the natural compound geodiamolide and the 18-membered ring
analog 129 with the numbering systems used.
1
H and 13C signal assignment was done by DQF-COSY, ROESY/NOESY, and HMQC spectra
in DMSO-d6 with results comparable to the 19-membered macrocycles: a single signal set for
129, the non-methylated amide and a doubled signal set for 134 with the cis isomer of the Nmethylated amide populated to less than 1%. Investigation of the NOESY/ROESY data results
in mean values of proton-proton distances being significant for the fast exchange of several
conformers. Thus the 18-membered rings 129 and 134 are more flexible than the systems
described above and a preferred structure can not be calculated on the basis of experimental
ROE data. ROESY data confirm a trans configuration for all amide bonds in 129, respectively
134. Additionally the ROESY data reveal again an oscillatory movement for the m-xylyl unit
like in 135 and 136. In case of the geodiamolide analogs 129 and 134 there are consistent
dynamics for the whole molecule but rather an independent dynamical behavior of the side
104
Results and Discussion
chains, i. e. benzyl- and methyl groups, as seen in ROESY spectra with cross signals of
different sign. The methylene groups in position 1 of 129 and in positions 1 and 10 of 134
exhibit one large and one small 3JHH coupling constant (Table 3) defining a preferred gaucheanti orientation whereas in position 6 the coupling constants possess mediated values upon the
presence of several rotamers. Temperature measurements yield low temperature coefficients
for NH4 and NH13 for both structures (NH4: -1.0 resp. –1.1 ppb K-1; NH13: +0.4 resp. –1.1
ppb K-1) with a high probability for those NH protons to take part in an intramolecular
hydrogen bond.
When calculating only a partial structure for 134 with NOESY/ROESY data a betaII'-turnlike structure is obtained for the macrocyclic part containing the phenylalanine unit. For this
case NH4 is part of a transannular hydrogen bond with CO(15) as partner, whereas for NH13
there is no geometrically reasonable arrangement for the formation of a hydrogen bond (Figure
4.3.4). At least for this section of the macrocyclic ring the ROESY data obtained are in
accordance with the distances typical for betaII ‘turns.
Angle
134 ideal βII turn
134
134
Figure 4.3.4: Comparison of the calculated partial structure of 134 with a betaII-turn
structure.
Nevertheless, the 18-membered rings 129 and 134 are more flexible compared to 135, 136 or
jasplakinolide and an equilibrium of several fast exchanging conformers is existent.
Results and Discussion
105
4.3.2 Conformational studies of geodiamolide analogues 159 and 160
1
HN
15
HO
O
13
N O
O
O
N 9
H
15
5
O
11
HN
3
HO
7
H
N
1
O
13
N O
3
O
5
O
11
7
N 9
H
160
159
Figure 4.3.5: Structures for the 17-membered ring analogs 159 and 160 with the numbering
systems used.
The major variation between the analogues 159 and 160 and the natural product
geodiamolide is the ring size, the former ones contain a 17-membered ring while the latter one
contains a 18-membered ring. Compound 159 is a depsipeptide while 160 is a macrolactam.
Homo- and heteronuclear signal assignments of 159 and 160 are based on DQF-COSY, HSQC
and HMBC spectra, respectively. Rotating-frame NOESY (ROESY) and NOESY spectra
corroborate the signal assignments. Each exhibits a single 1H and
13
C NMR signal set in
DMSO-d6 and the absence of NOE-contacts expected for cis-amide bonds approve the trans
configuration for all amide bonds in both macrocyclic rings 159 and 160. 3JHH and 2JHH
coupling constants are taken directly from the well-resolved 1H NMR spectra after LorentzGauss transformation. The temperature dependence of the NH chemical shifts yields
information about the solvent accessibility of the amide protons.[124-127] Values between –5.8
and –7.6 ppb K-1 exclude strong intramolecular hydrogen bonds in both molecules.
57 ROEs are observed for the lactone 159 and 45 ROEs for the lactam 160. The volume
integrals translate into average proton-proton distances according to published methods.[128]
The common motif Val-D-Tyr-Ala adopts a mainly extended conformation with interresidue
CαHi - NHi+1 average distances between 2.2 and 2.4 Å and intraresidue CαHi - NHi distances
approaching 3 Å. 1,3-allylic strain in the polypropionate moiety is documented by the strong
ROE contact 4H – 6H3 in both molecules. 3JHH coupling constants and ROEs allow the
106
Results and Discussion
prochiral assignment of the pro-R and pro-S protons of the methylene groups’ 3-CH2 and 7CH2 which form the flexible joints between both termini of the polypropionate unit and the
tripeptide unit. In both molecules, one of the 3JH3,H4 and one of the 3JH7,H8 coupling constants is
small (Table 4), by this proving that a main rotamer is adopted around the 3C-4C and the 7C8C bond in both molecules. Differences between 159 and 160 are detected only for the 3-CH2
which neighbours the lactone in 159 and the amide in 160, respectively. The rotamer with a
gauche-anti orientation is dominating in 160 and the gauche-gauche orientation in 159. The
18-CH2 protons are well resolved in the case of the lactone but form a higher-order spin
system in the case of the lactam. Chemical shift differences between compounds 1 and 2 are
restricted to the Northern half of the macrocyclic rings in the region of the Val residue.
Selected NOEs and 3J coupling constants served as weak distance and torsional restraints in
molecular dynamics simulations. The average structures represent minima on the potential
energy surface and the energy minimized structures are shown in Figure 4.3.6. Structural
differences are confined to the amino acid Val. The more folded structure of the lactone brings
13-NMe and 6-Me in closer contact which is documented by a relatively short long-range
NOE of 3.1 Å. The main difference between the ring-constrained analogs investigated here
and the parent macrolide geodiamolide[123] is an approximately 180° rotation of the propionate
relative to the tripeptide unit. As a consequence, the 6-Me group is oriented to the opposite
side of the macrocyclic rings which is documented by the intense transannular 13-NMe – 6Me NOE. The distance between these two groups is 7.7 Å in geodeamolide where they are
positioned on opposite ring sides (Figure 4.3.6). Such a strong effect of a ring contraction from
a 18-membered ring in geodiamolide to a 17-membered ring in 159 and 160 is completely
unexpected but well documented by the solution NMR data analyzed here.
Results and Discussion
107
Table 4: 2J and 3J coupling constants of 159and 160 in [Hz]
Coupling
159
160
JH3h,H3t
10.9 Hz
12.9 Hz
JH3h,H4
2.5 Hz
9.1 Hz
3
JH3t,H4
5.4 Hz
2.6 Hz
JH7h,H7t
15.1 Hz
16.0 Hz
<2 Hz
<2 Hz
11.1 Hz
≈10 Hz (COSY)
2
3
2
3
JH7h,H8
3
JH7t,H8
Figure 4.3.6: Energy minimized structures of compounds geodiamolide, 159, and 160.
108
Results and Discussion
Results and Discussion
109
Chapter II
Approach towards the Synthesis of the Stereotetrad of
Cruentaren-A
110
Results and Discussion
5 Introduction
Benzolactones may be viewed as privileged structures in nature since there are so
many of them and they do show a broad range of biological activity. They may be classified
according to their ring size, the substituents on the benzoic acid, and the nature of the side
chain that extends from the secondary alcohol function that is engaged in the lactone.
Salicylihalamide A (206) and apicularen A (207) are members of the so-called benzolactone
enamides (Figure 5.1).[130] These compounds possess an extended side chain with an enamide
functionality that is necessary for the biological activity. It is assumed that protonation of the
side chain generates an electrophilic acyliminium ion.[131] Many other benzolactones just have
a methyl substituent at the lactone. Zearalenone (208) is a fungal metabolite that exhibits
anabolic and antibacterial activity.[132] Among the benzolactones that shows promising
biological activities, cruentaren A (209) is a notable example.
H
N
OH
O
O
O
OH O
OH
O
O
O
N
H
OH apicularen A (207)
salicylihalamide A (206)
HO
O
HO
OH O
Me
OH
N
H
O
O
O
HO
zearalenone (208)
MeO
HO
cruentaren A (209)
Figure 5.1: Structures of some important benzolactones
Results and Discussion
111
The macrolide cruentaren A is a highly cytotoxic and antifungal natural product,
isolated from the strain Byssophaga cruenta by the Höfle research group in Braunschweig,
Germany. Structurally it belongs to the benzolactone family of natural products (Figure 5.1). It
features a 12-membered macrolactone ring and an allylamine side chain. Plus, there are some
stereocenters, a further typical feature of polyketides. Cruentaren A was originally patented as
a pesticide but in the meantime it turned out that it is a selective inhibitor of F-ATPase. But it
does not inhibit the V-ATPase which is the target of the benzolactone enamides. This
selectivity is quite impressive and synthesis of cruentaren analogues might provide some
information about the groups on cruentaren A that are responsible for this difference. One
might speculate that the allylamine rearranges to an enamide. It is interesting to note that there
are some other allylamine containing natural products, for example leucasandrolide and
ajudazol. Thus, synthetic chemistry will be necessary to clarify a range of questions posed by
the above benzolactones.
A possible retrosynthetic analysis for cruentaren A is shown in Scheme 66. It is based
on a Mitsunobu macrolactonization reaction. The seco-acid 210 would then be constructed
from alkyne 211 and epoxide 212. Other equivalents for alkyne 211 are of course conceivable.
On the other hand, compound 212 containing a stereotetrad seems to be an indispensable
intermediate, even for other strategies such as alkyne ring-closing metathesis.
112
Results and Discussion
HO
O
N
H
HO
OH
O
OH
CO2H
1. Mitsunobu macrolactonization
MeO
O
OH OPMB
OTBS
MeO
O
few steps
anti syn anti
HO
cruentaren A (209)
210
OH
CO2R
epoxide
opening
O
MeO
OPMB
OTBS
S
S
H
211
stereotetrad 212
Scheme 66: Retrosynthetic pathway to cruentaren A.
Here, in this work a total synthesis of cruentaren A is not proposed. Instead, our target
was an efficient synthesis of the stereotetrad 212.
6 Synthesis of stereotetrad 212
6.1.1 Retrosynthetic analysis of stereotetrad 212
From a retrosynthetic stand point one has to address the four chiral centers which are
arranged in anti-syn-anti fashion.[133] It would be better to synthesize the epoxide in the final
steps to avoid unnecessary side reactions. Thus, it was thought to start the synthesis from the
right side of the epoxide 212.
Results and Discussion
113
OPMB
O
7
5
OTBS
3
1
stereotetrad 212
Even though there are several accessible retrosynthetic analyses possible for the synthesis of
stereotetrad 212, it was decided to follow Scheme 67 which involves an Evans syn aldol
reaction[46] and a Sharpless dihydroxylation reaction[134] as key steps to create the stereo
centers on positions 4, 5 and 6, respectively.
OPMB
O
5
7
1. protection
1. Sharpless dihydroxylation
OTBS
3
1
OPMB
3. oxidation
2. epoxide
formation
O
O
OTBS
N
Bn
Evans syn
aldol
OH
O
O
O
O
OTBS
H
N
Bn
214
4. Wittig olefination
213
stereotetrad 212
O
2. reduction
OTBS
ent-100
215
Scheme 67: Retrosynthetic analysis for the compound 212
Epoxide 212 might be obtained from the corresponding olefin 213 in two steps using an
asymmetric Sharpless dihydroxylation followed by closing the epoxide. It should be noted that
the configuration of the hydroxyl group formed in the dihydroxylation could be analyzed by
forming the six-membered acetal. In turn, the olefin 213 would be obtained from the syn aldol
product 214 in few steps using protection, reduction, oxidation and Wittig olefination. The
aldehyde 215 could be achieved in few steps using asymmetric alkylation as the key step.
6.1.2 Synthesis of aldehyde 215
There are several methods known to prepare aldehyde 215.[135-138] The synthesis of the
aldehyde 215 followed almost the similar pathway as reported by Crimmins et al. [138] (Scheme
114
Results and Discussion
68). The synthesis began with alklylation of the Seebach auxiliary 216 with allyl bromide.[139]
After successfully achieving the alkylated product 217, the chiral auxiliary was removed using
NaBH4 in THF/H2O to get the alcohol 218 in almost quantitative yield. Subsequent protection
of the alcohol 218 using pivaloyl chloride in presence of diisopropylethylamine and a catalytic
amount of DMAP furnished the corresponding pivalate 219 in almost quantitative yield.
Dihydroxylation of pivalate 219 by in situ generated osmium tetroxide using
K2OsO4.2H2O/AcOH/H2O2[140] produced the diol which on oxidative cleavage using sodium
periodate gave the aldehyde which on reduction led to the alcohol 220. It should be noted that
compound 220 could be obtained also by ozonolysis of the alkene 219. Subsequent silylation
of the alcohol 220 using TBS-Cl in presence of imidazole produced the silylated alcohol 221
in almost quantitative yields, which was subjected to reductive removal of the pivaloyl group
using DIBAL-H at 0 oC leading to alcohol 222. The latter was subjected to Swern oxidation to
give aldehyde 215.
O
O
O
1. NaHDMS (2M sol in THF)
THF, -78 oC, 2 h
O
Ph Ph
O
N
2. allyl bromide, -78 oC to rt,
Ph Ph
90%
216
217
O
N
piv-Cl, DIEA, cat. DMAP
CH2Cl2, 100%
HO
NaBH4 in H2O, THF
85%
1. K2OsO4.2H2O, AcOH, H2O2
THF, H2O
PivO
2. NaIO4, THF/H2O (9:1)
218
OH
PivO
220
3. NaBH4, THF, 0 oC
219
1. TBS-Cl, imidazole,
DMF, rt, 90%
2. DIBAL-H, CH2Cl2,
0 oC, 90%
OTBS
HO
222
(COCl)2, DMSO
215
Et3N, CH2Cl2
-78 oC, 92%
Scheme 68: Synthesis of aldehyde 215.
After having successfully synthesized aldehyde 215, an aldol reaction was performed between
Evans reagent ent-100 and the aldehyde 215. However, a dibutylboron triflate mediated aldol
Results and Discussion
115
reaction between the Evans reagent ent-100 and the aldehyde 215 in presence of triethylamine
at -78 oC produced the aldol product 214 in very low yields. Attempts to increase the yield did
not give better results. The corresponding TiCl4/(-)-sparteine mediated aldol reaction did not
produce the product at all. At this point, we decided to follow a new strategy to synthesize the
compound 212 by avoiding the aldol reaction with aldehyde 215 as the production of this
aldehyde is costly and the aldol reaction might need an excess of aldehyde.
O
O
O
O
N
OTBS
H
ent-100 Bn
n-Bu2BOTf
DIPEA
CH2Cl2, -78 oC
22-25%
215
TiCl4
(-)-spartein
CH2Cl2, 0 oC
O
O
O
OH
OTBS
N
Bn
214
no reaction,
aldehyde got
decomposed
Scheme 69: Attempts to synthesis the compound 214
6.1.3 Second retrosynthetic pathway for epoxide 212
In the second retrosynthetic pathway we decided to synthesize the terminal hydroxyl group
after introducing the syn methyl and hydroxyl groups in the stereotetrad 212. In this plan, the
olefin 213 would originate from the alcohol 223 like in the last proposal (Scheme 70). The
alcohol 223 could be prepared from the reduction of PMP-acetal 224 using DIBAL-H. The
PMP-acetal 224 would be prepared from olefin 225 by dihydroxylation, oxidative cleavage,
reduction and protection of the resulting alcohol. The olefin 225 could be prepared from the
aldol product 226 by reduction and protection of the resulting diol with the p-methoxy
benzaldehyde acetal.
116
Results and Discussion
OPMB
OTBS
OH OPMB
223
1. dihydroxy
-lation
2. oxidative
cleavage
PMP
O
PMP
O
O
1. reduction
OTBS
2. PMP-acetal
protection
3. reduction
4. protection
224
O
O
1. DIBAL-H
OTBS
2. Wittig ole-fination
213
O
1. oxidation
225
O
OH
N
Evans syn
aldol
O
O
O
N
O
H
Bn
Bn
226
ent-100
227
Scheme 70: Second retrosynthetic pathway for the epoxide 212.
6.1.4 Synthetic pathway
The synthesis was started with the aldol reaction between the aldehyde 227 and the propionyl
oxazolidinone ent-100. The aldehyde 227 was prepared by a known procedure.[141] nButylboron triflate mediated aldol reaction in presence of the base triethylamine between the
aldehyde 227 and the chiral reagent ent-100 furnished the aldol product 226 in 72% yield with
good diastereoselectivity (Figure 6.1) as shown in Scheme 71. Subsequent reduction of the
aldol product 226 using NaBH4 in H2O gave the diol which was protected using p-methoxy
benzaldehyde dimethyl acetal in presence of catalytic amounts PPTS to furnish the PMPprotected olefin 225. The alkene 224, was then subjected to dihydroxylation using potassium
osmate dihydrate. The resulting diol then cleaved without further purification using sodium
periodate. However this process was a very messy reaction. There could be no product
observed in LC-MS.
Results and Discussion
O
O
O
N
H
227
PMP
O
O
n-Bu2BOTf,
Et3N
O
Bn
ent-100
117
O
CH2Cl2, -78 oC
75%
OR
1. NaBH4, THF/H2O
N
2. PMP-acetal, PPTS,
CH2Cl2
Bn
R = H 226
1. K2OsO4.2H2O, AcOH,
30 wt% H2O2, THF, tBuOH,
H2O
O
O
no aldehyde
2. NaIO4, THF/H2O (9:1)
225
Scheme 71: Attempts for the synthesis of alcohol 223
O
O
O
9.54
15.10
39.35
37.69
37.27
35.36
55.06
OH
N
Bn
150
66.10
77.32
77.00
76.68
74.64
116.36
136.93
134.96
129.36
128.90
127.37
152.80
177.86
Chloroform-d
226
100
50
ppm
Figure 6.1: 13C NMR spectrum of syn aldol product 226.
At this point, we decided not to introduce the PMP-acetal before oxidative cleavage of the
double bond. Thus, the aldol product was silylated using TBDMS-triflate in presence of 2,6lutidine to get the silylated ether 228, which was on reductive cleavage of the chiral auxiliary
118
Results and Discussion
using sodium borohydride led to the 1,6-diol 229 (Scheme 72). TBAF mediated cleavage of
the silyl ether in compound 229 produced the 1,3,6-triol 230 in 80% yield. Subsequent
protection of 1,3-diol subunit using p-methoxybenzaldehydedimethyl acetal in presence of
catalytic amounts of PPTS gave the 1,3 PMP-acetal product 231 in 75% yield. Protection of
the terminal alcoholic group in 231 provided the PMP-acetal product 224. Regioselective
reductive opening of the acetal 224 to PMB-alcohol 223 was achieved with DIBAL-H at 0 oC
in 75% yield. The resulting alcohol 223 was oxidized under Swern oxidation conditions
leading to the corresponding aldehyde 232 which was subjected to a Wittig olefination using
methyltriphenylphosphonium iodide (Ph3P+CH3I-) in presence of n-BuLi at 0 oC giving the
required olefin 213 72% yield. The synthesis of the epoxide 212 completely depends on the
selectivity of the Sharpless dihydroxylation step.[129] Even if the selectivity will be low, both
isomers might be separable. AD-mix-β mediated dihydroxylation of the olefin 213 produced
the dihydroxylated product 233 in 70% yield in 3 days at 0 oC with an approximate
diastereomeric ratio of 2:1 (determined from 13C NMR spectrum, Figure 6.2). Unfortunately,
the two diastereomers could not be separated on flash chromatography. AD-mix-α mediated
dihydroxylation produced a better selectivity 4:1 with opposite facial selectivity. In this case a
few milligrams (around 5 mg) of pure product could be isolated by flash chromatography. The
opposite facial selectivity could be explained using
13
C NMR spectra of both diastereomeric
mixtures as illustrated in Figure 6.2. The expanded region cleary showed the two different
chemical shifts for the methyl group at 5th position for both diastereomeric mixtures (8.6 ppm
for major diastereomer obtained using AD-mix-β and 11.4 ppm for major diastereomer
obtained using AD-mix-α). Due to the small amounts of diols the configurations could not
been designed definitely. At this point, one should note that one could in principle use the diol
mixture. After epoxide opening separation of the diastereomers might be possible. Using
appropriate reaction conditions for macrolactonization (Mitsunobu s. Yamaguchi) the two
isomers should converge to one product.
Results and Discussion
O
O
O
119
1. K2OsO4.2H2O, AcOH, H2O2
THF, H2O
OR
N
OH OR
PMP-acetal, PPTS,
OH
2. NaIO4, THF/H2O (9:1)
CH2Cl2, rt,
72%
o
3. NaBH4, THF/H2O, 20 C
Bn
80%
TBSOTf,
2,6-lutidine,
CH2Cl2, 90%
228 R = TBS
PMP
O
R = TBS 229
TBAF, THF
80%
226 R = H
R=H
230
PMP
O
O
TBDMS-Cl
OH
O
imidazole, DMF
rt, 85%
231
2. PPh3+CH3I- , n-BuLi
AD-mix-β,
tBuOH-H2O (1:1)
0 oC, 3 days
OPMB
OTBS
223
OH OPMB
HO
OTBS
70%
213
THF, 0 oC, 72%
OTBS
CH2Cl2, 0 oC
78%
224
1. (COCl)2, DMSO, Et3N,
CH2Cl2, -78 oC, 92%
OH OPMB
DIBAL-H
OTBS
233
AD-mix-α, tBuOH-H2O (1:1)
0 oC, 3 days
2:1 diastereomeric ratio
OH OPMB
HO
OTBS
diastereomer-233
4:1 diastereomeric ratio
Scheme 72: Synthetic pathway for epoxide 212.
epoxide 212
150
85
ppm
100
15
50
obtained by using AD-mix-β (upper) and AD-mix-α (lower).
-5.28
11.36
100
36.89
35.82
31.66
25.95
18.30
17.12
15
8.65
11.36
85
ppm
55.26
73.96
73.07
65.15
61.50
84.12
113.76
150
17.12
16.35
84.12
86.57
130.74
129.24
159.12
8.65
11.12
16.96
16.30
83.87
86.42
50
-5.32
36.93
36.76
35.81
35.37
31.72
31.64
18.27
16.96
16.30
11.12
8.65
86.42
83.87
75.19
73.89
73.13
72.88
65.13
65.04
61.49
61.30
113.80
130.77
130.36
129.25
159.15
159.04
120
Results and Discussion
Chloroform-d
ppm
10
ppm
0
Chloroform-d
ppm
10
ppm
0
Figure 6.2: 13C NMR spectra of dihydroxylation products 233 and diastereomer-233
Results and Discussion
121
The stereochemical outcome in the Sharpless dihydroxylation might be determined using the
Rychnovsky
13
C method.[142] Protection of the primary alcohol and removal of the PMB
protecting group would provide the 1,3-diol which on protection using 2,2-dimethoxypropane
in presence of CSA could provide the acetonide. It is known that the carbons of the methyl
groups in the acetonide of a 1,3-diol will exhibit different chemical shifts depending on the
relative stereochemistry. For example, in the case of a syn-1,3-diol acetonide, the two methyl
groups adopt different orientations (one is axial and the other is equatorial). So they have
different chemical shifts, and also the quarternery carbon typically appears at 98.5 ppm. In
case of an anti 1,3-diol, however, the acetonide will adopt a twist-boat form and the two
methyl groups become more equivalent. Therefore they have similar chemical shifts and the
quaternary carbon usually appears at 100.6 ppm.
R1
R2
R2
O
CH3
O
R1
O
30.0 ppm
O
HH
Chair
CH3
19.6 ppm
98.5 ppm
syn 1,3-diol
acetonide
R2
R1
O
O
anti 1,3-diol
acetonide
R2 H
1
R
H
O
CH3 24.6 ppm
O
CH3
24.6 ppm
100.6 ppm
Twist-boat
Figure 6.3: Rychnovsky 13C acetonide method for 1,3-diol stereo assignments.
122
Experimental Section
7 Summary and Conclusion
This dissertation contains two chapters. Chater I includes the efficient synthesis of rationally
designed amino- and hydroxy acids 95, 96, 97, 116, 194, 195 which incorporate
conformational constraints due to non bonded interactions such as syn-pentane and 1,3-allylic
strain. The design of the novel amino and hydroxy acids was guided by the polypropionate
sector 3 of the depsipeptide jasplakinolide.
R
95 R = H
CO2H 116 = Br
R = Boc
RHN
1,3-allylic interaction
RO
syn-pentane interaction
96
CO2H
R = TBDPS
simplify
HO
RHN
CO2H
97
3
RHN
CO2H
R = Boc
194
CO2H
R = Boc
195
CO2H
R = TBS
RO
Figure 7.1 Rational design of the novel ω-amino and hydroxy acids based on non bonded
interactions
The syntheses of these amino- and hydroxy acids were achieved in a few steps with good
yields. The amino acid 95 and 116 were synthesized in a similar pathway in six steps by using
a double alkylation, enzyme mediated hydrolysis of a homotopic diester and a Curtius
rearrangement as key steps. This way, the novel ω-amino acids 95 and 116 can be prepared in
gram quantities.
Summary and Conclusion
R
O
O
123
R
4 steps
O
N
Bn
HO
Br
Br
102
OMe
O
R = H 104
= Br 118
R = H 103
= Br 122
O
R
2 steps
NHBoc
HO2C
R = H 95
= Br 116
Scheme 73: Key substrates in the synthesis of novel amino acids 95 and 116
The amino acid 95 was incorporated into four macrolactams 129, 134, 135 and 136. The
former two feature a 18-membered ring whereas the latter two have a 19-membered ring.
OMe
Ph
H
Me
N
N
N
O
O
Me
N
R1
O
O
H
H
H
H
O
R1 = H 129
= CH3 134
N
N
N
N
O
O
Me
N
R2
H
H
O
R1 = H
135
= CH3 136
Figure 7.2: The structures of geodiamolide and jasplakinolide analogues.
For the two larger ones, conformational analysis showed that the macrocyclic rings are more
rigid than the jasplakinolide ring, but all in all their conformations are very well comparable to
the natural product. Like stated for jasplakinolide in the literature, the 19-membered analogs
exhibit neither intramolecular hydrogen bonds, nor is the cis-rotamer populated in case of Nmethylation (136). The conformational features of the 18-membered (geodiamolide) analogs
124
Summary and Conclusion
are quite different. The macrocyclic ring of compounds 129 and 134 is more flexible than the
other investigated systems. Due to the increased flexibility and signal overlap a distinct
solution structure for 129 and 134 could not be gained. Whether the ring size or the additional
aromatic side chains in 135 and 136 cause a stabilizing effect on the macrocyclic system could
not be determined. But both features are differing for 129 and 134 and might thus be an
explanation for the higher flexibility of these smaller macrocycles. In addition both Nmethylated analogs (134 and 136) populate the trans-amide conformer to more than 99%.
The novel hydroxy and amino acids 96 and 97 were obtained via a divergent synthesis starting
from an Evans aldol reaction. Reduction, mono protection, acylation, and an Ireland-Claisen
rearrangement provided the hydroxy acid 96 in good yields. Protection, hydrolysis, amidation,
reduction, acylation and an Ireland-Claisen rearrangement gave rise to the amino acid 97 with
excellent diastereoselectivity and good yields.
O
O
4 steps
O
H
96
O
O
N
1 step
OR
O
140 R = TBDPS
141
Bn ent-100
O
1 step
6 steps
97
O
NHBoc
147
Scheme 74: Key components in the synthesis of hydroxy- and amino acids 96 and 97.
The hydroxy- and amino acids were incorporated into a tripeptide to get a depsipeptide 159
and a macrolactam 160. The structural differences of 159 and 160 are confined to the amino
acid valine. The more folded structure of the lactone 159 brings the N-methyl and allylic
methyl group in closer contact which is documented by a relatively short range NOE of 3.1 Å.
The main difference between the ring-constrained analogues investigated here and the parent
macrolide geodiamolide is an approximately 180o rotation of the propionate relative to the
Summary and Conclusion
125
tripeptide unit. As a consequence, the allylic methyl group is oriented to the opposite side of
the macrocyclic rings which is documented by the intense transannular NMe and allylic
methyl NOE. The distance between these two groups is 7.7 Å in geodiamolide where they are
positioned on the opposite ring sides. Such a strong effect of a ring contraction from a 18membered ring in geodiamolide to a 17-membered ring in 159 and 160 is completely
unexpected but well documented by the solution NMR data. It should be noted that both
macrocycles populate the trans-amide conformer more than 99%.
O
HN
HO
N
O
O
O
O
N
H
159
H
N
HN
HO
N
O
O
O
O
N
H
160
Figure 7.3: Geodiamolide analogues containing the hydroxy- and amino acids 96 and 97.
The replacement of allylic system with the meta substituted aromatic ring in the above aminoand hydroxy acids 96 and 97 led to the design of the novel amino acids 194 and 195. The
amino acid 194 was prepared using the same starting materials as were used for the amino acid
95. Different reaction conditions allowed for a divergent synthesis and produced the amino
acid 195 in five steps with very good yields. The synthesis of hydroxy acid 195 was achieved
in only three steps using the same starting materials. The construction of macrocycles using
these acids is underway.
126
Summary and Conclusion
O
O
O
O
Br
N
Br
100 Bn
1 step
O
O
N
Bn
104
4 steps
Br
194
203
2 steps
195
Scheme 75: Key intermediates in the synthesis of amino-and hydroxy acids 194 and 195.
Nor methyl chondramide C 185 was efficiently synthesized by incorporating the hydoxy acid
96 into the tripeptide 183 as shown in Scheme 76. Normethyl chondramide C did show an
activity towards the L929-Mausfibroblast cells with an IC50 value of 0.25 μM (150 ng/mL).
The synthesis of the macrocycles described in this thesis reveals the fact that it is more easy to
form the amide bond rather the ester in order to close the ring.
OH
OTBS
O
O
H
H
N
Me
N
H
OR
N
H
HO
4 steps
O
O
N
Me
H
N
Boc
184 R = Me
tBuO
N
N
N
O
O
O
O
H
O
170
nor methyl chondramide C183
Scheme 76: Key components in the synthesis of nor methyl chondramide C.
The syntheses of amino- and hydroxy acids were achieved in a maximum five to six steps
using similar starting materials but building upon divergent syntheses. The main reactions
involved in the synthesis of these acids were the Evans asymmetric alkylation and aldol
reactions with propionyl oxazolidinone to create the chiral centers carrying methyl groups.
The novel amino and hydroxy acids could serve as novel workbenches for restricting the
Summary and Conclusion
127
conformation of small peptides. Furthermore, the aryl group might serve as a handle for
attachment of derived macrocycles to a solid surface. The incorporation of the m-xylene
subunit into the amino acid indicates that small structural modifications can have subtle effect
on a macrocyclic structure.
Chapter II describes efforts towards the synthesis of the stereotetrad 212 of cruentaren A
(209). The key steps in this synthesis were an asymmetric Evans syn aldol reaction and a
Sharpless dihydroxylation reaction. As the Sharpless dihydroxylation did not provide good
diastereoselectivity, it is necessary to follow some other pathway to create the
diastereomerically pure epoxide 212. But, it should be noted that this synthesis will become
efficient if both isomers could be separated from each other after the Sharpless
dihydroxylation step.
O
O
O
N
Bn
ent-100
O
5 steps
OH OTBS
4 steps
OH
H
215
O
2 steps
OH OPMB
OPMB
OTBS
OPMB
OTBS
OTBS
212
223
213
Scheme 76: Key intermediates in the synthesis of epoxide 212
128
Summary and Conclusion
8 Experimental Section
8.1 General Remarks
8.1.1 Chemicals and Working Techniques
The chemicals were purchased from the firms Acros, Aldrich, Fluka, Lancaster, Avocado and
Merck. All reagents were obtained from commercial suppliers, and were used without further
purification unless otherwise stated. All solvents were distilled and/or dried prior to use by
standard methodology except for those, which were reagent grades. The applied petroleum
ether fraction had a boiling point of 40-60 oC. Anhydrous solvents were obtained as follows:
THF, diethyl ether and toluene by distillation from sodium and benzophenone;
dichloromethane and chloroform by distillation from calcium hydride; acetone by distillation
from phosphorous pentoxide. Absolute triethylamine and pyridine and diisopropylethylamine
were distilled over calcium hydride prior to use. Unless and otherwise mentioned, all the
reactions were carried out under a nitrogen atmosphere and the reaction flasks were pre-dried
by heat gun under high vacuum. All the chemicals, which were air or water sensitive, were
stored under inert atmosphere. Compounds that are not described in the experimental part
were synthesized according to the literature.
8.1.2 NMR-spectroscopy
Except for the final compounds (600 MHz), all the spectra were measured on a Bruker
Advance 400 spectrometer, which operated at 400 MHz for 1H and 100 MHz for
respectively. 1H and
13
13
C nuclei,
C NMR spectra were performed in deuterated solvent and chemical
shifts were assigned by comparison with the residual proton and carbon resonance of the
solvent and tetramethylsilane (TMS) as an internal reference (δ = 0). Data are reported as
follows: chemical shift (multiplicity: s = singlet, d = doublet, t = triplet, ddd = doublet of
Experimental Section
129
doublet of doublet, dt = doublet of triplet, td = triplet of doublet, m = multiplet, br =
broadened, J = coupling constant (Hz), integration, peak assignment in italic form).
8.1.3 Mass Spectrometry
Mass spectra were recorded on a Finnigan Triple-Stage-Quadrupol Spectrometer (TSQ-70)
from Finnigan-Mat. High-resolution mass spectra were measured on a modified AMD Intectra
MAT 711 A from the same company. The used mass spectrometric ionization methods were
electron-impact (EI), fast-atom bombardment (FAB) or field desorption (FD). FT-ICR-mass
spectrometry and HR-FT-ICR mass spectra were measured on an APEX 2 spectrometer from
Bruker Daltonic with electrospray ionization method (ESI). Some of the mass spectra were
also measured on an Agilent 1100 series LC-MSD. Analytical HPLC-MS: HP 1100 Series
connected with an ESI MS detector Agilent G1946C, positive mode with fragmentor voltage
of 40 eV, column: Nucleosil 100–5, C-18 HD, 5 µm, 70 × 3 mm Machery Nagel, eluent: NaCl
solution (5 mM)/acetonitrile, gradient: 0/10/15/17/20 min with 20/80/80/99/99% acetonitrile,
flow: 0.6 mL min-1. High resolution mass (HRMS) are reported as follows: (ESI): calcd mass
for the related compound followed by found mass.
8.1.4 Infrared Spectroscopy
The FT-IR spectra were recorded on a Fourier Transform Infrared Spectrometer model Jasco
FT/IR-430. Solid samples were pulverized with potassium bromide and percent reflection
(R%) was measured. The percent transmittance (T%) of liquid substances were measured in
film between potassium bromide plates. Absorption band frequencies are reported in cm-1.
8.1.5 Polarimetry
Optical rotations were measured on a JASCO Polarimeter P-1020. They are reported as
follows: [α]temperatureD (concentration, solvent). The unit of c is g/100 mL. Anhydrous CH2Cl2
or CHCl3 was used as a solvent. For the measurement the sodium D line = 589 nm was used.
130
Experimental Section
8.1.6 Melting Points
Melting points were determined with a Büchi Melting point B-540 apparatus and were not
corrected.
8.1.7 Chromatographic Methods
Flash column chromatography was performed using flash silica gel (40-63 µm, 230-400 mesh
ASTM) from Macherey-Nagel.
Gas chromatography was performed on a CHROMPACK CP 9000 using a flame ionization
detector, and carrier gas H2. For GC-MS coupled chromatography, a GC-system series 6890
with an injector series 7683 and MS-detector series 5973 from Hewlett Packard was used, with
EI method, and carrier gas He. Analytical HPLC was performed on a Hewlett Packard HP
1100 system.
Analytical thin layer chromatography (TLC) was performed on precoated with silica gel 60
F254 plates (Merck) or Polygram Sil G/UV254 (Macherey Nagel). The compounds were
visualized by UV254 light and the chromatography plates were developed with an aqueous
solution of molybdophosphorous acid or an aqueous solution of potassium permanganate
(heating with the hot gun). For preparation of the molybdate solution 20 g ammonium
molybdate [(NH4)6Mo7O24.4H2O] and 0.4 g Ce(SO4)2.4H2O were dissolved in 400 mL of 10%
H2SO4. The potassium permanganate solution was prepared from 2.5 g KMnO4 and 12.5 g
Na2CO3 in 250 mL H2O.
8.1.8 Experimental procedures
All the experimental procedures are arranged in the ascending order of number of the
compound.
Experimental Section
131
(2S)-3-(3-{(2S)-2-[(tert-Butoxycarbonyl)amino]propyl}phenyl)-2-methylpropanoic acid
(95)
O
HO
NHBoc
NaOH (80 mg) in H2O (5 mL) was added to a stirred solution of methyl ester 103 (0.55 g, 1.64
mmol) in THF (12 mL). The reaction mixture was stirred for 14 h at room temperature before
being poured into water (25 mL) and extracted with diethyl ether (3 × 10 mL). The aqueous
layer was acidified to pH 2-3 with 1N HCl and extracted with ethyl acetate (3 × 15 mL). The
combined organic layers were dried (Na2SO4), filtered, and concentrated. The crude product
was purified by flash chromatography (1:3 ethyl acetate/petroleum ether) resulting in acid 95
as a white solid (0.44 g, 85%).
Rf = 0.45 (1:3 ethyl acetate/petroleum ether);
M.P. = 104-106 oC;
[α]23D = +6.01 (c 0.56, CH2Cl2);
IR (film): νmax = 3326, 2974, 2930, 1706, 1653, 1507, 1248, 1169 cm-1;
1
H NMR (400 MHz, CD3OD): δ = 0.94 (d, J = 6.1 Hz, 3H, CH3NHR), 0.99 (d, J = 6.6 Hz,
3H, CH3CO2H), 1.28 (s, 9H, C(CH3)3), 2.45-2.66 (m, 4H, benzylic H, CH), 2.86 (dd, J = 12.6,
6.1 Hz, 1H, benzylic H), 3.19 (s, 1H, NH), 3.61-3.66 (m, 1H, CHNH), 6.91-6.92 (m, 3H, aryl
H), 7.06 (t, J = 7.5 Hz, 1H, Hm, aryl H);
13
C NMR (100 MHz, CD3OD): δ = 17.2 (CH3CHNH), 20.5 (CH3CHCO2H), 28.8 (C(CH3)3),
40.7 (CH2CHCO2H), 42.7 (CHCO2H), 43.8 (CH2CHNH), 49.5 (CHNH), 79.8 (Boc C), 127.9,
128.3, 129.2, 131.2, 140.3, 140.7 (aryl), 157.7 (Boc C=O), 179.9 (CO2H);
HRMS (ESI): calcd for C18H27NO4 [M+Na]+ 344.18323, found 344.18313.
132
Experimental Section
(2S,4E,6S)-7-{[tert-Butyl(diphenyl)silyl]oxy}-2,4,6-trimethylhept-4-enoic acid (96)
HO2C
OTBDPS
To a solution of diisopropyl amine (0.20 mL, 1.40 mmol) in dry THF (2 mL) was added nBuLi (2.5 M solution in hexane) (0.56 mL, 1.40 mmol) at 0 oC. Stirring was continued for 30
min at 0 oC. HMPA (0.5 mL) was added and the reaction mixture cooled to -78 oC. Ester 140
(500 mg, 1.17 mmol) in dry THF (0.3 mL) was added dropwise to the above reaction mixture
and after stirring for an hour at -78 oC, TBDMS-Cl (265 mg, 1.76 mmol) in THF (0.6 mL) was
added dropwise. After 30 min stirring at -78 oC, the cooling bath was removed and reaction
mixture brought to room temp and heated at 60 oC for 10 h. The reaction mixture was cooled
to room temperature and treated with saturated NH4Cl (5 mL) and then diluted with 1N HCl (5
mL), followed by stirring for 5 min. The mixture was extracted with ethyl acetate (3 x 10 mL).
The combined ethyl acetate layers were washed with brine, dried (Na2SO4), filtered, and
concentrated in vacuo to give the crude product which was purified by flash chromatography
(1:3 ethyl acetate/petroleum ether) resulting in pure hydroxy acid 96 (360 mg, 72%) as a
colorless gel.
Rf = 0.45 (1:3 ethyl acetate/petroleum ether);
[α]20D = +4.3 (c 1.13, CH2Cl2);
IR (film): υmax = 3448, 2966, 2879, 1718, 1629, 1439, 1377, 1195, 1159, 1103, 1053 cm-1;
1
H-NMR (400 MHz, CDCl3): δ = 0.94 (d, J = 6.8 Hz, 3H, CH3CHCH2O), 1.04 (s, 9H,
C(CH3)3), 1.09 (d, J = 7.1 Hz, 3H, CH3CHCO), 1.56 (s, 3H, CH3), 2.02 (dd, J = 13.4, 8.1 Hz,
CH2CO2), 2.37 (dd, J = 13.3, 6.7 Hz, CH2CO2), 2.54-2.63 (m, 2H, CH, CH), 3.41-3.49 (m, 2H,
CH2OH), 4.99 (d, J = 9.1 Hz, 1H, olefin H), 7.36-7.43 (m, 6H, aromatic), 7.82 (d, J = 6.8 Hz,
4H, aromatic);
13
C NMR (100 MHz, CDCl3): δ = 15.8 (CH3), 16.2 (CH3CHCO), 17.3 (CH3CHCH2O), 19.2
(quarternery), 26.8 (3C, tBu), 35.4 (CHCO2), 37.8 (CHCH2O), 43.8 (CH2CO), 68.5 (CH2O),
127.6, 129.5 (aromatic), 130.8 (olefinic), 132.0 (aromatic), 134.0 (olefin quarternary), 135.6
(aromatic), 182.8 (CO2H);
HRMS (EI): calcd for C26H36O3Si [M+Na]+: 447.23259, found 447.23264.
Experimental Section
133
(2S,4E,6S)-7-[(tert-Butoxycarbonyl)amino]-2,4,6-trimethylhept-4-enoic acid (97)
HO2C
NHBoc
To solution of ester 147 (140 mg, 0.49 mmol) in THF / HMPA (1.0/0.3 mL) was added a 2 M
solution of NaHDMS in THF (1.5 mL) at -78 oC. After stirring for 45 min at -78 oC, TMS-Cl
(0.5 mL, 4.00 mmol) and Et3N (0.20 mL, 1.50 mmol) were added simultaneously. After 15
min, the cooling bath was removed and the reaction mixture was allowed to come to room
temperature in 1 h. The mixture was heated at 60 oC for 5 h. The reaction mixture was treated
with saturated aq. NH4Cl (2 mL) and diluted with 1N HCl (2 mL), then extracted with ethyl
acetatae (3 x 5 mL). The combined organic layers washed with brine (4 mL), dried (Na2SO4),
filtered, and concentrated in vacuo to give the crude amino acid which was purified by flash
chromatography (1:1 ethyl acetate/petroleum ether). This way the pure amino acid 97 (90 mg,
65%) was obtained as a colorless oil.
Rf = 0.45 (1:1 ethyl acetate/petroleum ether);
[α]20D = -34.6 (c 0.62, CH2Cl2);
IR (film): υmax = 3361, 2974, 2930, 1735, 1712, 1511, 1172 cm-1;
1
H NMR (400 MHz, CDCl3): δ = 0.89 (d, J = 6.6 Hz, 3H, CH3NHBoc), 1.12 (d, J = 6.8 Hz,
3H, CH3CHCO), 1.42 (s, 9H, tBu), 1.62 (s, 3H, CH3C=C), 2.03-2.10 (m, 1H, CH2CHCO),
2.34-2.39 (m, 1H, CH2CHCO), 2.56-2.67 (m, 2H, CHNH, CHCO), 2.76-2.78 (m, 1H,
CH2NH), 3.11-3.14 (m, 1H, CH2NH), 4.55 (broad s, 1H, NH), 4.91 (d, J = 9.4 Hz, 1H, olefinic
CH);
13
C NMR (100 MHz, CDCl3): δ = 16.1 (CH3C=C), 16.3 (CH3CHCO), 18.2 (CH3CHCH2NH),
28.4 (3C, tBu), 33.0 (CHCH2NH), 37.9 (CHCO), 45.4 (CH2CHCO), 46.4 (CH2NH), 79.1 (Boc
quarternary), 130.8 (CH olefinic), 133.4 (olefin quarternary), 156.00 (Boc C=O), 181.9 (acid
C=O);
HRMS (EI): calcd for C15H27NO4 [M+Na]+: 308.18323, found 308.18317.
134
Experimental Section
(2S)-3-{3-[(2S)-2-Carboxypropyl]phenyl}-2-methylpropanoic acid (98)
O
HO
O
OH
To a solution of the bisalkylated compound 108 (7.50 g, 13.2 mmol) in THF (250 mL), H2O2
(11.7 mL of a 30 wt% solution, 102.7 mmol) was added at 0 °C through a syringe, followed by
the addition of lithium hydroxide monohydrate (2.20 g, 51.4 mmol), dissolved in water (120
mL). The solution was stirred at 0 °C for 5 h. Subsequently, saturated Na2SO3 solution (100
mL) and saturated NaHCO3 solution (100 mL) were added at 0 °C. The whole mixture was
partially concentrated in vacuo and diluted with water (100 mL). The aqueous layer was
extracted with dichloromethane (3 × 75 mL) to recover the auxiliary. The aqueous layer was
then acidified at 0 °C to pH 1.5 using 6M HCl and then extracted with ethyl acetate (4 × 100
mL). The combined ethyl acetate layers were dried (MgSO4), filtered, and concentrated in
vacuo yielding a colorless oily residue 98 (2.95 g, 90%).
[α]25D = +35.5 (c 0.42, CH2Cl2);
IR (film): νmax = 3500-2500 (broad), 1702, 1589, 1463, 1292, 1199, 1044 cm-1;
1
H NMR (400 MHz, CDCl3): δ = 1.20 (d, J = 6.8 Hz, 6H, CH3), 2.62-2.72 (m, 2H, benzylic
H), 2.75-2.83 (m, 2H, CH), 3.10 (dd, J = 13.1, 6.1 Hz, 2H, benzylic H), 7.07 (d, J = 7.1 Hz,
aryl H), 7.10 (s, 1H, Ho, aryl H), 7.26 (t, J = 7.6 Hz, 1H, Hm, aryl H), 11.79 (broad, 2H,
CO2H);
13
C NMR (400 MHz, CDCl3): δ = 16.3 (CH3), 39.1 (benzylic), 41.3 (CH), 127.1, 128.4, 129.7,
139.0 (aryl), 182.7 (CO2H);
HRMS (EI): calcd for C14H18O4 [M]+: 250.12049, found 250.118291.
Experimental Section
135
(2S)-3-{3-[(2S)-3-Methoxy-2-methyl-3-oxopropyl]phenyl}-2-methylpropanoic acid (103)
O
MeO
O
OH
A solution of diester 109 (2.50 g, 9.0 mmol) in MeOH (5 mL) was emulsified under vigorous
stirring in NaCl solution (0.1 M, 646.75 mL) to which pH 7 phosphate buffer (3.25 mL) was
added, making the solution 3 mM in phosphate. Then a suspension of PLE (25 mg, 1000 units,
Sigma Aldrich, E-3019) in 3.2 M (NH4)2SO4 solution (1 mL) was added. During the
hydrolysis the pH was kept between 7 and 7.5 by the controlled addition of NaOH solution
(0.1 N). After observing the formation of diacid in HPLC-MS (maximum 12 h), the reaction
mixture was washed with CH2Cl2 (2 × 500 mL). The aqueous phase was acidified to pH 2.5
with 25% hydrochloric acid and extracted with ethyl acetate (3 × 500 mL). The combined
organic layers (CH2Cl2 and EtOAc) were dried over Na2SO4, filtered, and concentrated in
vacuo. The crude product was purified by flash chromatography (1:4 ethyl acetate/petroleum
ether) to provide the mono acid mono ester 103 (1.52 g, 64%) as a colorless oily compound.
Rf = 0.4 (1:4 ethylacetate/petroleum ether,)
[α]25D = +46.2 (c 1.07, CH2Cl2);
IR (film): νmax = 3024, 2975, 1736, 1707 cm-1;
1
H NMR (400 MHz, CDCl3): δ = 1.05 (d, J = 6.8 Hz, 3H, CH3CHCO2CH3), 1.08 (d, J = 7.1
Hz, 3H, CH3CHCO2H), 2.53-2.58 (m, 2H, benzylic H), 2.62-2.68 (m, 2H, CH), 2.94 (ddd, J =
19.4, 13.1, 6.4 Hz, 2H, benzylic H), 3.55 (s, 3H, OCH3), 6.90-6.96 (m, 3H, aryl H), 7.12 (t, J =
7.5 Hz, 1H, Hm, aryl H), 10.49(broad, 1H, CO2H);
13
C NMR (100 MHz, CDCl3): δ = 16.4 (CH3CO2CH3), 16.6 (CH3CO2H), 39.1
(CH2CHCO2CH3), 39.6 (CH2CHCO2H), 41.4 (CHCO2H), 51.5 (OCH3), 126.9, 127.0, 128.4,
129.8, 139.0 (aryl), 176.4 (CO2Me), 182.3 (CO2H);
HRMS (EI): calcd for C15H20O4 [M]+: 264.133877, found 264.136141.
136
Experimental Section
(4R)-4-Benzyl-3-[(2S)-3-(3-{(2S)-3-[(4R)-4-benzyl-2-oxo-1,3-oxazolidin-3-yl]-2-methyl-3oxopropyl}phenyl)-2-methylpropanoyl]-1,3-oxazolidin-2-one (108)
O
O
O
N
Bn
O
O
N
O
Bn
To a solution of diisopropylamine (8.0 mL, 56.2 mmol) in THF (190 mL) was added nbutyllithium (22.5 mL, 56.2 mmol, 2.5 M in hexane) at 0 °C. The reaction mixture was stirred
at 0 °C for 30 min, before it was cooled to -78 °C. At this point propionyl oxazolidinone 100
(12.0 g, 51.5 mmol), dissolved in THF (210 mL) was added. After being stirred for 1.5 h at –
78 °C, the solid 1,3-bis-(bromomethyl)-benzene (104) (6.17 g, 23.4 mmol) was added in one
portion. Stirring was continued for 24 h with simultaneous warming of the reaction mixture to
room temperature. The reaction was quenched with 60 mL of NH4Cl, and then most of the
organic solvent was removed in vacuo. The remainder was extracted with ethyl acetate (3 × 50
mL) and the combined organic layers were washed with brine, dried with Na2SO4, filtered and
concentrated in vacuo. The residue was purified by flash chromatography (3:7 ethyl
acetate/petroleum ether) to give 108 as a sticky compound (7.72 g, 58%).
Rf = 0.45 (3:7 ethyl acetate/petroleum ether);
[α]25D = -21.9 (c 1.50, CH2Cl2);
IR (film): νmax = 3028, 2976, 2932, 1770, 1694, 1604, 1588, 1487, 1455, 1393, 1288, 1210,
1103, 1053 cm-1;
1
H NMR (400 MHz, CDCl3): δ = 1.18 (d, J = 6.6 Hz, 6H, CH3), 2.54-2.69 (m, 4H, benzylic
H), 3.11-3.18 (m, 4H, PhCH2), 4.10-4.20 (m, 6H, CHCH3, OCH2), 4.64-4.70 (m, 2H, NCH),
7.09-7.32 (m, 14H, aryl H);
13
C NMR (100 MHz, CDCl3): δ = 16.2 (CH3), 37.3 (PhCH2), 39.1 (CHCH3), 39.9 (benzylic),
54.7 (NCH), 65.5 (OCH2), 126.8, 127.9, 128.5, 129.0, 130.1, 134.9, 138.9 (aryl), 152.6
(NCO2), 176.1 (CO);
HRMS (EI): calcd for C34H36N2O6 [M]+: 568.257798, found 568.257289.
Experimental Section
137
Methyl (2S)-3-{3-[(2S)-3-Methoxy-2-methyl-3-oxopropyl]phenyl}-2-methylpropanoate
(109)
O
O
MeO
OMe
To a solution of diacid 98 (3.20 g, 12.8 mmol), methanol (1.4 mL, 32.8 mmol) and DMAP (96
mg) in dry CH2Cl2 (37 mL) was added at 0 °C a solution of DCC (8.12 g, 39.4 mmol) in
CH2Cl2 (26 mL). The solution was stirred at 0 °C for 30 min and then at room temperature for
6 h. The white precipitate was filtered off, the solvent was evaporated, and the residue was
redissolved in diethyl ether. The ether solution was washed successively with cold 1 N HCl,
NaHCO3 solution, and brine. The dried (Na2SO4) ether layer was filtered, and concentrated.
The crude product was purified by flash chromatography (1:9 ethyl acetate/petroleum ether) to
provide the diester 109 as an oily compound (2.52 g, 71%).
Rf = 0.38 (1:9 ethyl acetate/petroleum ether);
[α]25D = +40.1 (c 0.61, CH2Cl2);
IR (film): νmax = 2974, 2951, 1736, 1459, 1375, 1361, 1164 cm-1;
1
H NMR (400 MHz, CDCl3): δ = 1.15 (d, J = 6.5 Hz, 6H, CH3), 2.61-2.67 (m, 2H, benzylic
H), 2.69-2.77 (m, 2H, CH), 3.00 (dd, J = 13.1, 6.7 Hz, 2H, benzylic H), 3.64 (s, 6H, OCH3),
6.97 (s, 1H, Ho, aryl H), 7.01 (d, J = 7.6 Hz, 2H, aryl H), 7.20 (t, J = 7.6 Hz, 1H, Hm, aryl H);
13
C NMR (100 MHz, CDCl3): δ = 16.6 (CH3), 39.6 (benzylic), 41.6 (CH), 51.5 (OCH3),
126.9, 128.3, 129.6, 139.3 (aryl), 176.4 (CO2Me);
HRMS (EI): calcd for C16H22O4 [M]+: 278.15179, found: 278.152648
138
Experimental Section
Methyl (2S)-3-(3-{(2S)-2-[(tert-Butoxycarbonyl)amino]propyl}phenyl)-2methylpropanoate (115)
O
MeO
NHBoc
A solution of the monoester 103 (0.80 g, 3.03 mmol) in toluene (23 mL) was treated with
triethylamine (0.5 mL, 3.33 mmol) and DPPA (0.66 mL, 3.03 mmol). After stirring for 30
min, the mixture was heated to reflux for 3.5 h. The isocyanate formation was monitered by IR
for the appearance of a strong signal in the 2300-2200 cm-1 region and disappearance of the
carboxylic acid carbonyl peak. The reaction mixture was cooled to 50 °C, tert-butanol (3 mL,
10 eq) was added via syringe and the solution heated to reflux for 20 h. The reaction was
cooled to room temperature and quenched with saturated NaHCO3 solution (25 mL). The
mixture was extracted with diethyl ether (3 × 25 mL). The combined ether extracts were dried
(Na2SO4), filtered, and concentrated in vacuo. The crude product was purified by flash
chromatogrophy (1:5 ethyl acetate/petroleum ether), to provide compound 115 as an oil (0.73
g, 72%).
Rf = 0.55 (1:5 ethyl acetate/petroleum ether);
[α]23D = +12.3 (c 0.21, CH2Cl2);
IR (film): νmax = 3361, 2973, 2930, 2359, 1735, 1710, 15516, 1364, 1166 cm-1;
1
H NMR (400 MHz, CDCl3): δ = 0.99 (d, J = 6.6 Hz, 3H, CH3CHCO2Me), 1.06 (d, J = 6.6
Hz, 3H, CH3CHNR), 1.36 (s, 9H, C(CH3)3) 2.51-2.58 (m, 2H, benzylic H), 2.63-2.68 (m, 1H,
CHCO2Me), 2.75 (dd, J = 13.1, 5.3 Hz, 1H, benzylic H), 2.93 (dd, J = 13.1, 6.4 Hz, 1H,
benzylic H), 3.57 (s, 3H, OCH3), 3.81 (br s, 1H, CHNHR), 4.31 (br s, 1H, NH), 6.90-7.32 (m,
4H, aryl H);
13
C NMR (100 MHz, CDCl3): δ = 16.3 (CH3CHCO2Me), 19.7 (CH3CHNHR), 28.0
(C(CH3)3)), 39.2 (CH2CHCO2Me), 41.0 (CHCO2Me), 42.5 (CH2CHNHR), 47.1 (CHNHR),
51.2 (OCH3), 78.8 (Boc quaternary C), 126.6, 127.2, 127.9, 129.9, 137.9, 139.0 (aryl), 154.8
(Boc CO), 176.2 (CO2Me);
HRMS (EI): calcd for C19H29NO4 [M]+: 335.209636, found 335.207186.
Experimental Section
139
(2S)-3-(3-Bromo-5-{(2S)-2-[(tert-butoxycarbonyl)amino]propyl}phenyl)-2methylpropanoic acid (116)
Br
BocHN
CO2H
The procedure for amino acid 95 was used with methyl ester 123 (130 mg, 0.31 mmol) in THF
(2.5 mL) and NaOH (20 mg) in H2O (1.0 mL) to yield 100 mg (80%) of the bromo amino acid
116 after prurification by flash chromatography (1:3 ethyl acetate/petroleum ether) as a
colorless gel.
Rf = 0.41 (1:3 ethyl acetate/petroleum ether);
[α]23D = +11.0 (c 1.00, CH2Cl2);
IR (film): νmax = 3326, 2974, 2930, 1706, 1653, 1507, 1248, 1169 cm-1;
1
H NMR (400 MHz, CD3OD): δ = 1.06 (d, J = 6.3 Hz, 3H, CH3NHR), 1.11 (d, J = 6.8 Hz,
3H, CH3CO2H), 1.37 (s, 9H, C(CH3)3), 2.58-2.70 (m, 4H, benzylic H, CH), 2.93 (dd, J = 12.9,
6.8 Hz, 1H, benzylic H), 3.72-3.78 (m, 1H, CHNH), 4.96 (br s, 1H, CHNH), 7.00 (s, 1H, aryl
H), 7.19 (s, 1H, aryl H), 7.20 (s, 1H, aryl H);
13
C NMR (100 MHz, CD3OD): δ = 17.2 (CH3CHNH), 20.6 (CH3CHCO2H), 28.8 (C(CH3)3),
40.1 (CH2CHCO2H), 42.3 (CHCO2H), 43.3 (CH2CHNH), 48.8 (CHNH), 79.8 (Boc C), 122.9,
130.1, 130.7, 131.3, 142.6, 143.0 (aryl), 157.5 (Boc C=O), 179.4 (CO2H);
HRMS (ESI): calcd for C18H26BrNO4 [M+Na]+ 422.09374, found 422.09422.
(4R)-4-Benzyl-3-[(2S)-3-(3-bromo-5-{(2S)-3-[(4R)-4-benzyl-2-oxo-1,3-oxazolidin-3-yl]-2methyl-3-oxopropyl}phenyl)-2-methylpropanoyl]-1,3-oxazolidin-2-one (119)
O
O
O
O
O
N
N
Bn
Bn
Br
O
140
Experimental Section
The alkylation procedure (for the compound 108) was used with propionyl oxazolidinone 100
(4.35 g, 18.30 mmol) in THF (90 mL) and the tribromo derivative 118 (3.00 g, 8.75 mmol),
diisopropylamine (3.00 mL, 21.00 mmol), and n-BuLi (8.40 mL, 2.5 M in hexane, 21.00
mmol) to yield 3.00 g (53%) of double alkylation product 119 after flash chromatography (1:3
ethyl acetate/petroleum ether) as a light yellow gel.
Rf = 0.38 (1:3 ethyl acetate/petroleum ether);
[α]20D = -11.4 (c 2.11, CH2Cl2);
IR (film): νmax = 3028, 2976, 2932, 1770, 1694, 1604, 1588, 1487, 1455, 1393, 1288, 1210,
1103, 1053 cm-1;
1
H NMR (400 MHz, CDCl3): δ = 1.09 (d, J = 6.8 Hz, 6H, CH3), 2.49-2.55 (m, 4H, benzylic
H), 3.01-3.10 (m, 4H, PhCH2), 3.94-3.99 (m, 2H, CHCH3), 4.01-4.07 (m, 2H, OCH2), 4.10 (t,
J = 8.5 Hz, 2H, OCH2), 4.56-4.62 (m, 2H, NCH), 7.02 (d, J = 6.6 Hz, 4H, aryl), 7.09 (s, 1H,
aryl), 7.18-7.24 (m, 8H, aryl);
13
C NMR (100 MHz, CDCl3): δ = 16.6 (CH3), 37.7 (PhCH2), 39.2 (CHCH3), 39.5 (benzylic),
55.1 (NCH), 65.9 (OCH2), 122.2, 127.3, 128.9, 129.3, 130.2, 135.2, 141.5 (aryl), 153.0
(NCO2), 176.0 (CO);
HRMS (EI): calcd for C34H35BrN2O6 [M+Na]+: 671.15707, found 671.15644.
(2S)-3-{3-Bromo-5-[(2S)-2-carboxypropyl]phenyl}-2-methylpropanoic acid (120)
Br
HO2C
CO2H
The procedure for diacid 98 was used with the double alkylated product 119 (2.50 g, 3.86
mmol) in THF (80 mL), 30 wt% H2O2 (3.15 mL, 30.88 mmol) and lithium hydroxide
monohydrate (650 mg, 15.44 mmol) in H2O (30 mL) to provide 1.13 g of diacid 120 (89%)
after workup. The crude product was used without any purification.
[α]20D = +40.5 (c 1.00, CH2Cl2);
IR (film): νmax = 3500-2500 (br), 1700, 1585, 1465, 1292, 1199, 1044 cm-1;
Experimental Section
1
141
H NMR (400 MHz, CDCl3): δ = 1.15 (d, J = 6.8 Hz, 6H, CH3), 2.64 (dd, J = 12.9, 7.1 Hz,
2H, benzylic H), 2.68-2.75 (m, 2H, CH), 2.92 (dd, J = 12.9, 6.6 Hz, 2H, benzylic H), 6.92 (s,
1H, aryl), 7.17 (s, 2H, aryl);
13
C NMR (400 MHz, CDCl3): δ = 16.5 (CH3), 38.9 (benzylic), 41.1 (CH), 122.3, 128.4, 130.2,
141.1 (aryl), 181.4 (CO2H);
HRMS (EI): calcd for C14H17BrO4 [M-H]-: 327.02375, found 327.02366.
Methyl (2S)-3-{3-bromo-5-[(2S)-3-methoxy-2-methyl-3-oxopropyl]phenyl}-2methylpropanoate (121)
Br
MeO2C
CO2Me
The procedure for diester 109 was used with diacid 120 (1.10 g, 3.34 mmol) in
dichloromethane (10 mL) and DCC 1.73 g, 8.35 mmol) in dichloromethane (8.0 mL), DMAP
(245 mg, 2.00 mmol) to yield the diester 121 (895 mg, 75%) after purification by flash
chromatography (1:9 ethyl acetate/petroleum ether) as a colorless oil.
Rf = 0.44 (1:9 ethyl acetate/petroleum ether);
[α]20D = +52.4 (c 1.05, CH2Cl2) ;
IR (film): νmax = 2974, 2951, 1736, 1459, 1375, 1361, 1164 cm-1;
1
H NMR (400 MHz, CDCl3): δ = 1.07 (d, J = 7.1 Hz, 6H, CH3), 2.53 (dd, J = 13.3, 7.5 Hz,
2H, benzylic H), 2.59-2.7 (m, 2H, CH), 2.88 (dd, J = 13.3, 7.0 Hz, 2H, benzylic H), 3.57 (s,
6H, OCH3), 6.82 (s, 1H, aryl), 7.09 (s, 2H, aryl);
13
C NMR (100 MHz, CDCl3): δ = 16.6 (CH3), 39.0 (benzylic), 41.1 (CH), 51.4 (OCH3),
122.1, 128.3, 129.8, 141.4 (aryl), 175.8 (CO2Me);
HRMS (EI): calcd for C16H21BrO4 [M]+: 379.05154, found, 379.05137.
142
Experimental Section
(2S)-3-{3-Bromo-5-[(2S)-3-methoxy-2-methyl-3-oxopropyl]phenyl}-2-methylpropanoic
acid (122)
Br
MeO2C
CO2H
A solution of diester 121 (820 mg, 2.23 mmol) in MeOH (1.25 mL) was emulsified under
vigorous stirring in NaCl solution (0.1 M, 160.0 mL) to which pH 7 phosphate buffer (0.80
mL) was added, making the solution 3 mM in phosphate. Then a suspension of PLE (12 mg,
240 units, Sigma Aldrich, E-3019) in 3.2 M (NH4)2SO4 solution (0.25 mL) was added. During
the hydrolysis the pH was kept between 7 and 7.5 by the controlled addition of NaOH solution
(0.1 N). After observing the formation of diacid in LC-MS (7-8 h), the reaction mixture was
washed with CH2Cl2 (2 x 200 mL). The aqueous phase was acidified to pH 2.5 with 25%
hydrochloric acid and extracted with ethyl acetate (3 x 200 mL). The combined organic layers
(CH2Cl2 and EtOAc) were dried over Na2SO4, filtered, and concentrated in vacuo. The crude
product was purified by flash chromatography (1:4 ethyl acetate/petroleum ether) to provide
the mono acid mono ester 122 (435 mg, 57%) as a colorless oily compound.
Rf = 0.44 (1:4 ethyl acetate/petroleum ether);
[α]20D = +48.4 (c 0.98, CH2Cl2);
IR (film): νmax = 3020, 2970, 1735, 1707 cm-1;
1
H NMR (400 MHz, CDCl3): δ = 1.12 (d, J = 6.8 Hz, 3H, CH3CHCO2CH3), 1.15 (d, J = 7.1
Hz, 3H, CH3CHCO2H), 2.58 (dd, J = 13.4, 7.8 Hz, 2H, benzylic H), 2.64-2.75 (m, 2H, CH),
2.93 (dd, J = 12.4, 6.1 Hz, 1H, benzylic H), 2.98 (dd, J = 12.6, 5.8 Hz, 1H, benzylic H), 3.62
(s, 3H, OCH3), 6.89 (s, 1H, aryl H), 7.15 (s, 1H, aryl H), 7.17 (s, 1H, aryl H), 9.62 (br, 1H,
CO2H);
13
C NMR (100 MHz, CDCl3): δ = 16.5 (CH3CO2CH3), 16.7 (CH3CO2H), 38.7
(CH2CHCO2CH3), 39.1 (CH2CHCO2H), 41.0 (CHCO2CH3), 41.2 (CHCO2H), 51.7 (OCH3),
122.3, 128.5, 130.0, 130.1, 141.2, 141.6 (aryl), 176.2 (CO2Me), 181.7 (CO2H);
HRMS (EI): calcd for C15H19BrO4 [M-H]-: 341.03940, found 341.03930.
Experimental Section
143
Methyl (2S)-3-(3-bromo-5-{(2S)-2-[(tert-butoxycarbonyl)amino]propyl}phenyl)-2methylpropanoate (123)
Br
MeO2C
NHBoc
To a stirred solution of monoester mono acid 122 (320 mg, 0.93 mmol) in tBuOH (10 mL)
were added triethylamine (0.15 mL, 1.02 mmol) and DPPA (0.20 mL, 0.93 mmol)
successively at room temperature. After stirring for 0.5 h at room temperature the reaction
mixture was heated to reflux for 10 h. The reaction mixture was treated with saturated aq.
NaHCO3 (10 mL). The resulting mixture was extracted with diethyl ether (3 x 10 mL). The
combined ether layers were dried (Na2SO4), filtered and concentrated in vacuo. The crude
product was purified by flash chromatography (1:5 ethyl acetate/petroleum ether) to produce
the corresponding Boc carbamate 123 (210 mg, 55%) as a colorless oil.
Rf = 0.52 (1:5 ethyl acetate/petroleum ether);
[α]20D = +12.3 (c 1.05, CH2Cl2);
IR (film): νmax = 3361, 2973, 2930, 2359, 1735, 1710, 15516, 1364, 1166 cm-1;
1
H NMR (400 MHz, CDCl3): δ = 0.99 (d, J = 6.8 Hz, 3H, CH3CHCO2Me), 1.07 (d, J = 7.1
Hz, 3H, CH3CHNR), 1.36 (s, 9H, C(CH3)3) 2.48-2.55 (m, 2H, benzylic H), 2.59-2.68 (m, 1H,
CHCO2Me), 2.73 (dd, J = 13.0, 3.9 Hz, 1H, benzylic H), 2.90 (dd, J = 13.4, 6.8 Hz, 1H,
benzylic H), 3.58 (s, 3H, OCH3), 3.78 (br s, 1H, CHNHR), 4.30 (br s, 1H, NH), 6.83 (s, 1H,
aryl), 7.10 (s, 2H, aryl);
13
C NMR (100 MHz, CDCl3): δ = 16.7 (CH3CHCO2Me), 20.0 (CH3CHNHR), 28.3
(C(CH3)3)), 39.1 (CH2CHCO2Me), 41.2 (CHCO2Me), 42.4 (CH2CHNHR), 47.4 (CHNHR),
51.7 (OCH3), 79.2 (Boc quaternary C), 122.1, 126.1, 128.9, 129.9, 140.5, 141.6 (aryl), 155.1
(Boc CO), 176.1 (CO2Me);
HRMS (EI): calcd for C19H28BrNO4 [M+Na]+: 436.10939, found 436.10826.
144
Experimental Section
Methyl N-(tert-butoxycarbonyl)-D-alanyl-L-phenylalaninate (126)
Ph
H
CO2Me
N
O
H
N
Boc
To a stirred solution of N-Boc-D-alanine (125) (1.10 g, 5.6 mmol), L-phenylalanine
methylester (124) (1.00 g, 5.6 mmol), hydroxybenzotriazole (0.75 g, 5.6 mmol) in dry THF
(45 mL) was added a solution of DCC (1.5 g, 7.25 mmol) in THF (11 mL) at 0 °C. Stirring
was continued for 12 h at room temperature. The dicyclohexylurea was filtered off, washed
with cold diethyl ether and the filtrate was concentrated. Purification was done by flash
chromatography (1:3 ethyl acetate/petroleum ether) yielding a colorless solid 126 (1.55 g,
80%).
Rf = 0.35 (1:3 ethyl acetate/petroleum ether);
M.P. = 98-99 oC;
[α]23D = +59.8 (c 0.40, CH2Cl2);
IR (KBr): νmax = 3304, 2987, 2930, 1732, 1664, 1517, 1317 cm-1;
1
H NMR (400 MHz, CDCl3): δ = 1.21 (d, J = 7.1 Hz, 3H, CH3), 1.36 (s, 9H, C(CH3)3), 3.00
(dd, J = 13.8, 6.2 Hz, 1H, benzylic H), 3.05-3.10 (m, 1H, benzylic H), 4.04-4.18 (m, 1H,
CHNH), 4.77-4.81 (m, 1H, CHCO2Me), 4.95 (br s, 1H, NHBoc), 6.60 (br s, 1H,
NHCHCO2Me), 7.03-7.22 (m, 5H, aryl H);
13
C NMR (100 MHz, CDCl3): δ = 18.4 (CH3), 28.2 (C(CH3)3), 37.8 (benzylic), 49.9
(CHCO2Me), 52.3 (OCH3), 53.0 (CHNHBoc), 80.0 (Boc C), 127.1, 128.5, 129.2, 135.7 (aryl),
155.3 (Boc C=O), 171.7 (CO2Me), 172.2 (CONH);
HRMS (EI): calcd for C14H18N2O5 [M-C(CH3)3]+: 294.119319, found 294.121541.
Experimental Section
145
Methyl N-(tert-Butoxycarbonyl)-L-alanyl-D-alanyl-L-phenylalaninate (127)
Ph
H
Me
H
N
CO2Me
N
O
O
N
H
Boc
A solution of peptide 126 (1.0 g, 2.8 mmol) in CH2Cl2 (22 mL) was treated with CF3CO2H
(2.2 mL) and the mixture stirred at room temperature for 1 h. The solvent was removed in
vacuo, and the residue dried by azeotropic removal of H2O with toluene. The crude material
was subjected to the next reaction without further purification. To a cooled (0 °C) solution of
the crude amine salt (240 mg, 0.96 mmol) and N-Boc-L-alanine (181 mg, 0.96 mmol) in THF
(11 mL) and CH2Cl2 (2.5 mL) were added 1-hydroxybenzotriazole (130 mg, 0.96 mmol), Et3N
(0.32 mL, 2.3 mmol) and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride
(EDCI) (239 mg, 1.25 mmol). The mixture was then stirred at room temperature for 16 h. The
solvent was removed in vacuo, and the residue purified by flash chromatography (2:3 ethyl
acetate/petroleum ether) to provide the tripeptide 127 as a colorless solid (0.30 g, 75%).
Rf = 0.33 (2:3 ethyl acetate/petroleum ether);
M.P = 149-151 oC;
[α]28D = +19.1 (c 0.4796, CH2Cl2);
IR (KBr): νmax = 3277, 2979, 2748, 1753, 1707, 1645, 1519, 1456, 1370, 1166 cm-1;
1
H NMR (400 MHz, CDCl3): δ = 1.19 (d, J = 7.1 Hz, 3H, CH3CHNHBoc), 1.24 (d, J = 7.0
Hz, 3H, CH3CONH), 1.36 (s, 9H, C(CH3)3), 2.96 (dd, J = 13.9, 7.1 Hz, 1H, benzylic H), 3.073.12 (m, 1H, benzylic H), 3.62 (s, 3H, OCH3), 4.02-4.17 (m, 1H, CHNHBoc), 4.40-4.47 (m,
1H, CHNHCO), 4.73-4.78 (m, 1H, CHCO2Me), 5.11 (d, J = 7.3 Hz, 1H, NHBoc), 6.84 (d, J =
7.3 Hz, 1H, NHCO2Me), 6.95 (d, J = 6.6 Hz, 1H, NHCO), 7.05-7.22 (m, 5H, aryl H);
13
C NMR (100 MHz, CDCl3): δ = 18.1 (CH3CHNHBoc), 18.4 (CH3CHCONH), 28.3
C(CH3)3), 37.8 (benzylic), 48.5 (CHCONH), 50.2 (CHNHBoc), 53.2 (CHCO2Me), 80.1 (Boc
146
Experimental Section
C), 127.0, 128.5, 129.2, 135.8 (aryl), 155.3 (Boc C=O), 171.7 (CO2Me), 171.8 (NHCO), 172.6
(CH2NHCO);
HRMS (EI): calcd for C21H31N3O6 [M]+: 421.221247, found 421.225247.
Methyl N-[(2S)-3-(3-{(2S)-2-[(tert-butoxycarbonyl)amino]propyl}phenyl)-2methylpropanoyl]-L-alanyl-D-alanyl-L-phenylalaninate (128)
Ph
H
Me
H
N
CO2Me
N
NHBoc
O
O
N
H
O
123
A solution of peptide 127 (100 mg, 0.24 mmol) in CH2Cl2 (2 mL) was treated with CF3CO2H
(0.18 mL), and the mixture stirred at room temperature for 1 h. The solvent was removed in
vacuo, and the residue was dried by azeotropic removal of H2O with toluene. The crude
material was subjected to the next reaction without further purification. To a solution of crude
amine salt and Boc-protected ω-amino acid 95 (77 mg, 0.24 mmol) in dimethyl formamide (2
mL) were added TBTU (77 mg, 0.24 mmol), HOBt (34 mg, 0.24 mmol), DIEA (0.1mL, 0.576
mmol) and the mixture was stirred for 3 h at room temperature. The reaction mixture was
diluted with water (3 mL) and extracted with ethyl acetate (3 × 4 mL). The combined ethyl
acetate layers were washed with water resulting in almost pure tetrapeptide 128 (133 mg,
90%) as judged by HPLC-MS. This material was used for the macrolactam formation without
any further purification.
Experimental Section
147
(3S,6S,9R,12S,15S)-6-Benzyl-3,9,12,15-tetramethyl-4,7,10,13-tetraaza
bicyclo[15.3.1]henicosa-1(21),17,19-triene-5,8,11,14-tetrone (129)
Ph
H
Me
O
N
N
N
O
O
Me
N
H
H
H
O
NaOH (7.7 mg), dissolved in H2O (0.5 mL) was added to a stirred solution of tetrapeptide 128
(100 mg, 0.160 mmol) in THF (3 mL). The reaction mixture was stirred for 1 h at room
temperature before being poured into water (5 mL) and extracted with diethyl ether (3 × 5
mL). The aqueous layer was acidified to pH 2-3 with 1N HCl and extracted with ethyl acetate
(3 × 5 mL). The combined organic layers were dried (Na2SO4), filtered, and evaporated
providing the carboxylic acid in almost quantitative yield. This compound was used directly in
the next step. A solution of above Boc-protected tetrapeptide acid (90 mg, 0.145 mmol) in
CH2Cl2 (1.2 mL) was treated with CF3CO2H (0.11 mL, 1.45 mmol), and the mixture stirred at
room temperature for 1 h. The solvent was removed in vacuo, and the residue was dried by
azeotropic removal of H2O with toluene. The crude material was subjected to the
macrolactamization without any further purification. Thus, the residue was dissolved in DMF
(140 mL) and the stirred solution treated successively with TBTU (140 mg, 0.435 mmol),
HOBt (59 mg, 0.435 mmol), and iPr2EtN (0.08 mL, 0.44 mmol) at room temperature. The
resulting solution was stirred for 14 h at room temperature and then partitioned between ethyl
acetate and water. After separation of the layers, the water layer was extracted with ethyl
acetate (2 × 75 mL), and the combined organic layers were washed successively with water,
5% aqueous KHSO4, water, half-saturated NaHCO3, and brine. After being dried (MgSO4),
filtered, and concentrated in vacuo, the resulting residue was purified by flash chromatography
(ethyl acetate) to give the macrocycle 129 as a colorless solid (40 mg, 50%, 3 steps).
Rf = 0.5 (ethyl acetate);
148
Experimental Section
M.P. = 258-260 oC;
[α]28D = -14.8 (c 0.1911, CH2Cl2);
IR (film): νmax = 3297, 2931, 1888, 1640,1526, 1446 cm-1;
1
H NMR (600 MHz, DMSO-d6): δ = 1.01 (d, J = 7.3 Hz, 5-CH3), 1.06 (d, J = 6.6 Hz, 3H, 11-
CH3), 1.11 (d, J = 7.3 Hz, 3H, 5-CH3), 1.12 (d, J = 6.6 Hz, 3H, 14-CH3), 2.50 (m, 2H, 6-H, 11H), 2.59-2.67 (m, 2H, 10-H), 2.86-2.95 (m, 2H, 1-H, 6-H), 3.33 (dd, J = 13.9, 3.7 Hz, 1H, 1H), 3.74-3.79 (m, 1H, 17-H), 3.94-4.03 (m, 3H, 2-H, 14-H, 5-H), 6.75-6.79 (m, 2H, 4’’-H, 6’’H), 6.83 (d, J = 7.3 Hz, 2H, 4-NH, 13-NH), 6.94 (s, 1H, 2’’-H), 6.98 (dd, J = 7.3, 7.3 Hz, 1H,
5’’-H), 7.15-7.19 (m, 3H, 4’-H, aryl H), 7.23-7.27 (m, 2H, aryl H), 8.17 (d, J = 8.1 Hz, 1H,
19-NH), 8.34 (br s, 1H, 16-NH);
13
C NMR (150 MHz, DMSO-d6): δ = 15.8 (CH3-17), 17.9 (CH3-11), 18.3, 19.1 (CH3-5, CH3-
14), 34.6 (C-1), 39.1 (C-6), 39.5 (C-10), 42.0 (C-11), 45.0 (C-5), 47.6 (C-14), 49.6 (C-17),
54.4 (C-2);
HRMS (EI): calcd for C28H36N4O4 [M]+: 492.273621, found 492.271404.
Methyl N-(tert-butoxycarbonyl)-N-methyl-D-alanyl-L-phenylalaninate (131)
Ph
H
CO2Me
N
O
Me
N
Boc
To a stirred solution of N-Boc-N-methyl-D-alanine (130) (1.5 g, 7.4 mmol), L-phenylalanine
methylester (124) (1.3 g, 7.4 mmol), and hydroxybenzotriazole (0.99 g, 7.4 mmol) in dry THF
(60 mL) was added a solution of DCC (2.3 g, 11.1 mmol), dissolved in THF (11 mL) at 0 °C.
Stirring was continued for 7 h at room temperature. The dicyclohexylurea was filtered off,
washed with cold diethyl ether and the filtrate concentrated. Purification of the residue by
flash chromatography (1:5 ethyl acetate/petroleum ether) gave a colorless gel-like compound
131 (2.10 g, 75%).
Experimental Section
149
Rf = 0.33 (1:5 ethyl acetate/petroleum ether);
[α]25D = +62.8 (c 0.89, CH2Cl2);
IR (film): νmax = 3327, 2977, 2934, 2118, 1746, 1688, 1515, 1455, 1390, 1154 cm-1;
1
H NMR (400 MHz, CDCl3): δ = 1.23 (d, J = 6.8 Hz, 3H, CH3), 1.37 (s, 9H, C(CH3)3), 2.64
(s, 3H, NCH3), 2.99-3.07 (m, 2H, benzylic H), 3.63 (s, 3H, OCH3), 4.71-4.77 (m, 2H,
CHCO2Me, CHMe), 6.50 (br s, 1H, NHCHCO2Me), 7.03 (d, J = 7.3 Hz, 2H, Ho, aryl H), 7.157.23 (m, 3H, aryl H);
13
C NMR (100 MHz, CDCl3): δ = 13.2 (CH3), 28.3 (C(CH3)3), 29.7 (NCH3), 37.8 (benzylic),
52.2 (OCH3, CHCO2Me), 52.9 (CHNHBoc), 80.5 (Boc C), 127.1, 128.6, 129.1, 135.7, 171.3
(CO2Me), 171.7 (CONH);
HRMS (EI): calcd for C19H28N2O5 [M]+: 364.199791, found 364.198514.
Methyl N-(tert-Butoxycarbonyl)-L-alanyl-N-methyl-D-alanyl-L-phenylalaninate (132)
Ph
H
Me
Me
N
CO2Me
N
O
O
N
H
Boc
A solution of peptide 131 (1.0 g, 2.8 mmol) in CH2Cl2 (22 mL) was treated with CF3CO2H
(2.2 mL), and the mixture was stirred at room temperature for 1 h. The solvent was removed in
vacuo, and the residue dried by azeotropic removal of H2O with toluene. The crude material
was subjected to the next reaction without further purification. To a stirred solution of the
crude amine salt, N-Boc-alanine (0.53 g, 2.8 mmol), and PyBroP (1.3 g, 2.8 mmol) in CH2Cl2
(3 mL) was added DIPEA (1.4 mL, 8.4 mmol) at 0 °C and the mixture stirred at room
temperature for 3 h. The solvent was removed in vacuo and the residue purified by flash
chromatography (1:1 ethyl acetate/petroleum ether) to provide a gel-like compound 132 (0.69
g, 58%).
150
Experimental Section
Rf = 0.45 (1:1 ethyl acetate/petroleum ether);
[α]25D = +62.4 (c 1.79, CH2Cl2);
IR (film): νmax = 3327, 2979, 1742, 1682, 1642, 1520, 1455, 1249, 1169 cm-1;
1
H NMR (400 MHz, CDCl3): δ = 1.21 (d, J = 6.8 Hz, 3H, CH3CHNCH3), 1.23 (d, J = 7.8 Hz,
3H, CH3CHNHBoc), 1.37 (s, 9H, C(CH3)3), 2.85 (s, 3H, NCH3), 2.97 (dd, J = 13.9, 7.1 Hz,
1H, benzylic H), 3.04-3.09 (m, 1H, benzylic H), 3.60 (s, 3H, OCH3), 4.44-4.50 (m, 1H,
CHNHBoc), 4.65-4.70 (m, 1H, CHCO2Me), 5.11 (q, J = 6.3 Hz, 1H, CHNCH3), 5.31 (d, J =
6.8 Hz, 1H, NHBoc), 6.70 (d, J = 7.8 Hz, 1H, NHCHCO2Me), 7.05 (d, J =7.3 Hz, 2H, Ho, aryl
H), 7.14-7.24 (m, 3H, aryl H);
13
C NMR (100 MHz, CDCl3): δ = 13.5 (CH3CHCO), 17.9 (CH3CHNHBoc), 28.2 (C(CH3)3),
30.3 (NCH3), 37.4 (benzylic), 46.6 (CHNHBoc), 52.1 (OCH3, CHNMe), 53.0 (CHCO2Me),
79.6 (Boc C), 127.0, 128.4, 129.0, 135.9 (aryl) 155.3 (Boc C=O), 170.4 (CO2Me), 171.7
(CONMe), 173.7 (CONH);
HRMS (EI): calcd for C22H33N3O6 [M]+: 435.236897, found 435.240391.
Methyl N-[(2S)-3-(3-{(2S)-2-[(tert-butoxycarbonyl)amino]propyl}phenyl)-2methylpropanoyl]-L-alanyl N-methyl-D-alanyl-L-phenylalaninate (133)
Ph
H
Me
Me
N
CO2Me
N
NHBoc
O
O
N
H
O
A solution of tripeptide 132 (180 mg, 0.413 mmol) in CH2Cl2 (3.5 mL) was treated with
CF3CO2H (0.32 mL), and the mixture stirred at room temperature for 1 h. The solvent was
removed in vacuo, and the residue dried by azeotropic removal of H2O with toluene. The
crude material was subjected to the next reaction without further purification. To a cooled (0
°C) solution of the crude amine salt and amino acid 95 (133 mg, 0.413 mmol) in THF (7 mL)
Experimental Section
151
and CH2Cl2 (1.5 mL) were added 1-hydroxybenzotriazole (56.2 mg, 0.413 mmol), Et3N (0.15
mL, 1.03 mmol) and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (103 mg,
0.54 mmol), followed by stirring of the mixture at room temperature for 16 h. The solvent was
removed in vacuo, and the residue purified by flash chromatography (ethyl acetate/petroleum
ether, 1:1) to provide a colorless gel-like compound 133 (0.14 g, 55%).
Rf = 0.33 (1:1 ethylacetate/petroleum ether);
[α]25D = +61.5 (c 0.52, CH2Cl2);
IR (film): νmax = 3315, 2975, 2932, 1742, 1644, 1526, 1455, 1391, 1247, 1172 cm-1;
1
H NMR (400 MHz, CD3OD): δ = 1.04 (d, J = 6.6 Hz, 6H, CH3CHNH, CH2CH(CH3)), 1.23
(d, J = 7.3 Hz, 3H, CH3CHN(CH3)), 1.30 (d, J = 7.1 Hz, 3H, CH3CHNHBoc), 1.36 (s, 9H,
C(CH3)3), 2.40-2.72 (m, 4H, benzylic H), 2.86 (s, 3H, NCH3), 3.09-3.16 (m, 2H, PhCH2), 3.58
(s, 3H, OCH3), 3.68 (m, 1H, NHBoc), 4.50-4.60 (m, 2H, CH(CH3)NH, CH(CH3)NHBoc), 5.03
(q, J = 7.1 Hz, 1H, CH(CH3)NCH3), 6.89-7.20 (m, 11H, aryl H, PhCH2CHNH), 7.90 (br s, 1H,
COCHNH);
13
C NMR (100 MHz, CD3OD): δ = 14.0 (CH3CHN(CH3)), 16.7 (CH2CH(CH3)), 17.4
(CH3CHNH), 20.6 (CH3CHNHBoc), 28.8 (C(CH3)3), 31.4 (NCH3), 38.1 (PhCH2), 38.9
(CH(CH3)CO), 40.7 (CH2CH(CH3)CO), 43.0 (CH2CH(CH3)NH), 47.3 (CH(CH3)NH), 52.7
(CH(CH3)NCH3), 53.8 (OCH3), 55.5 (CHCO2Me), 79.8 (Boc C), 127.8, 128.0, 128.3, 129.2,
129.4, 130.2, 138.4, 140.4, 140.8 (aryl), 157.7 (Boc C=O), 173.1 (N(CH3)CO), 173.4
(CO2Me), 175.2 (COCHN(CH3)), 178.7 (NHCOCH);
HRMS (EI): calcd for C35H50N4O7 [M+Na]+: 661.35717, found 661.34712.
152
Experimental Section
(3S,6S,9R,12S,15S)–6–Benzyl-3,9,10,12,1–pentamethyl-4,7,10,13tetraazabicyclo[15.3.1]henicosa-1,17,19–trien-5,8,11,14–tetrone (134)
Ph
H
Me
O
N
N
N
O
O
Me
N
Me
H
H
O
NaOH (7.5 mg) in H2O (0.5 mL) was added to a stirred solution of tetrapeptide 133 (100 mg,
0.156 mmol) in THF (3 mL). The reaction mixture was stirred for 1 h at room temperature
before being poured into saturated NaHCO3 solution (5 mL) and extracted with diethyl ether
(3 × 5 mL). The aqueous layer was acidified to pH 2-3 with 1N HCl and extracted with ethyl
acetate (3 × 5 mL). The combined organic layers were dried (Na2SO4), filtered, and
concentrated in vacuo to give the free acid in almost quantitative yield. This acid was used for
the next step without further purification. To a solution of above N-Boc protected tetrapeptide
acid (90 mg, 0.144 mmol) in CH2Cl2 (1.2 mL) was added CF3CO2H (0.11 mL, 1.45 mmol),
and the mixture was stirred at room temperature for 1 h. The solvent was removed in vacuo,
and the residue was dried by azeotropic removal of H2O with toluene. The crude material was
subjected to the macrolactamization without any further purification. The residue was
dissolved in DMF (140 mL) and the stirred solution was treated successively with TBTU (140
mg, 0.435 mmol), HOBt (59 mg, 0.435 mmol), and iPr2NEt(0.08 mL, 0.435 mmol) at room
temperature. The solution was stirred at room temperature for 14 h and then partitioned
between ethyl acetate and water. After separation of the layers, the water was layer extracted
with ethyl acetate (2 × 75 mL). The combined organic layers were washed successively with
water, 5% aqueous KHSO4, water, half-saturated NaHCO3 and brine, dried (MgSO4), filtered,
and concentrated in vacuo. The residue was purified by flash chromatography (ethyl acetate)
to provide a colorless sticky solid 134 (50 mg, 62%, 3 steps).
Rf = 0.55 (ethyl acetate);
Experimental Section
153
[α]28D = +18.0 (c 0.30, CH2Cl2);
IR (film): νmax = 3302, 2933, 1633, 1520, 1455 cm-1;
1
H NMR (600 MHz, DMSO-d6): δ = 0.97 (d, J = 7.3 Hz, 17-CH3), 1.07 (d, J = 7.3 Hz, 3H,
CH3), 1.09 (d, J = 5.9 Hz, 3H, 11-CH3), 1.10 (d, J = 5.9 Hz, 3H, CH3), 2.36-2.43 (m, 1H, 11H), 2.48 (m, 1H, 10-H), 2.49 (m, 1H, 6-H), 2.60-2.70 (m, 2H, 10-H, 1-H), 2.84 (s, 3H, NCH3),
2.95 (dd, J = 13.2, 3.7 Hz, 1H, 6-H), 3.35 (dd, J = 13.6, 3.3 Hz, 1H, 1-H), 4.04-4.08 (m, 1H, 5H), 4.22-4.29 (m, 2H, 17-H, 2-H), 4.39-4.44 (m, 1H, 14-H), 6.46 (br s, 3H, 13-NH), 6.50 (d, J
= 8.1 Hz, 4-NH), 6.65, 6.70 (2 d, J = 7.3 Hz, 2H, 4’’-H, 6’’-H), 6.90 (dd, J = 7.3, 7.3 Hz, 1H,
5’’-H), 6.93 (s, 1H, 2’’-H), 7.15-7.19 (m, 1H, 4’-H), 7.21-7.27 (m, 4H, 2’-H, 4’-H), 8.31 (d, J
= 8.8 Hz, 1H, 19-NH);
13
C NMR (150 MHz, DMSO-d6): δ = 14.3 (CH3-17), 17.5 (CH3-11), 18.4 (CH3-5, CH3-14),
30.5 (NCH3), 35.4 (C-1), 38.6 (C-6), 40.5 (C-10), 43.6 (C-11), 44.3 (C-5), 44.7 (C-14), 53.3
(C-17), 53.4 (C-2);
HRMS (ESI): calcd for C29H38N4O4 [M+Na]+: 529.27853, found 529.27827.
(1S)-1-((1R)-2-{[tert-Butyl(diphenyl)silyl]oxy}-1-methylethyl)-2-methylprop-2-enyl
propionate (140)
O
O
OTBDPS
To a solution of alcohol 144 (500 mg, 1.36 mmol) in dry CH2Cl2 (5 mL) were added pyridine
(0.22 mL, 2.68 mmol) and n-propionyl chloride (0.18 mL, 2.00 mmol) at 0 oC. The reaction
mixture was warmed to room temperature and stirred for 12 h at room temperature. The
reaction mixture was diluted with CH2Cl2 (4 mL) and washed with brine, dried (Na2SO4),
filtered and concentrated in vacuo to give the crude product which was purified by flash
chromatography (5:95 ethyl acetate/petroleum ether) resulting in the acylated product 140
(500 mg, 87%) as a colorless gel.
Rf = 0.55 (5:95 ethyl acetate/petroleum ether);
154
Experimental Section
[α]20D = -11.0 (c 1.02 CH2Cl2);
IR (film): υmax = 3066, 2938, 2865, 1739, 1519, 1461, 1095 cm-1;
1
H NMR (400 MHz, CDCl3): δ = 0.87 (d, J = 6.8 Hz, 3H, CH3CH), 1.05 (s, 9H, tBu), 1.12 (t,
J = 7.6 Hz, 3H, CH2CH3), 1.67 (s, 3H, CH3C=CH), 1.96-2.02 (m, 1H, CHCH2O), 2.31 (q, J =
7.6 Hz, 3H, CH2CH3), 3.48-3.51 (m, 2H, CH2O), 4.84 (s, 1H, olefinic), 4.87 (s, 1H, olefinic),
5.34 (d, J = 5.1 Hz, 1H, CHO), 7.35-7.42 (m, 6H, aromatic), 7.64 (t, J = 5.1 Hz, 4H,
aromatic);
13
C NMR (100 MHz, CDCl3): δ = 9.2 (CH2CH3), 11.2 (CH3CH), 19.0 (CH3C=C), 19.2
(quarternary TBDPS), 26.8 (3C, tBu), 27.7 (CH2CH3), 37.7 (CHCH3), 65.4 (CH2OH), 76.5
(CHOH), 112.0 (olefinic CH2), 127.6, 129.6, 133.6, 133.7, 135.6, 135.6 (aromatic), 142.3
(olefinic quarternary);
HRMS (EI): calcd for C26H36O3Si [M+Na]+: 447.23259, found 447.23264.
(2R,3S)-2,4-Dimethylpent-4-ene-1,3-diol (143)
OH
OH
To a solution of aldol product 142 (1.00 g, 3.30 mmol) in THF (90 mL) was added NaBH4
(620 mg, 16.50 mmol) in H2O (20 mL) at 0 oC. The mixture was stirred for 7 h at room
temperature. The reaction mixture was treated with saturated aq. NH4Cl (20 mL) and stirred
for 1 h at room temperature. After separation of the layers, the aqueous layer was extracted
with ethyl acetate (3 x 50 mL). The combined organic layers were washed with saturated aq.
NaHCO3 (50 mL), brine (50 mL), dried (Na2SO4), filtered and concentrated in vacuo to give
the
crude
product
which
was
purified
by
flash
chromatoghaphy
ethylacetate/dichloromethane) to obtain the pure diol 143 (365 mg, 85%) as colorless oil.
Rf = 0.23 (5:95 ethyl acetate/dichloromethane);
[α]20D = -14.2 (c 1.02, CH2Cl2);
IR (film): υmax = 3370, 2965, 2931, 1446, 1095 cm-1;
(5:95
Experimental Section
1
155
H-NMR (400 MHz, CDCl3): δ = 0.86 (d, J = 6.8 Hz, 3H, CH3CH), 1.69 (s, 3H, CH3C=C),
1.84-1.89 (m, 1H, CHCH2O), 2.51 (br s, 1H, OH), 2.60 (br s, 1H, OH), 3.63-3.71 (m, 2H,
CH2O), 4.22 (br s, 1H, CHO), 4.91 (s, 1H, olefinic), 4.93 (s, 1H, olefinic);
13
C-NMR (100 MHz, CDCl3): δ = 9.8 (CH3CH), 19.4 (CH3C=C), 37.2 (CHCH3), 66.8
(CH2OH), 77.4 (CHOH), 110.6 (olefinic CH2), 146.3 (olefinic quarternary);
(3S,4R)-5-{[tert-Butyl(diphenyl)silyl]oxy}-2,4-dimethylpent-1-en-3-ol (144)
OH
OTBDPS
To a stirred solution of alcohol 143 (300 mg, 2.30 mmol) in dry DMF (10 mL), were added
imidazole (392 mg, 5.75 mmol) and TBDPS-Cl (380 mg (0.65 mL), 2.53 mmol) successively
at room temperature. Stirring was continued for 12 h. Then the reaction mixture was diluted
with water (10 mL) and stirred for 0.5 h before the mixture was extracted with diethyl ether (3
x 15 mL). The combined ether layers were washed with 1 N HCl (10 mL), saturated aq.
NaHCO3 (10 mL), brine (15 mL), dried (MgSO4), filtered and concentrated in vacuo to furnish
the crude product which was purified by flash chromatography (5:95 ethyl acetate/petroleum
ether) giving the mono protected alcohol 144 (870 mg, 97%) as a colorless gel compound.
Rf = 0.24 (5:95 ethyl acetate/petroleum ether);
[α]20D = -6.5 (c 1.00, CH2Cl2);
IR (film): υmax = 3370, 2965, 2931, 1446, 1095 cm-1;
1
H NMR (400 MHz, CDCl3): δ = 0.87 (d, J = 6.8 Hz, 3H, CH3CH), 1.06 (s, 9H, tBu), 1.66 (s,
3H, CH3C=CH), 1.83-1.90 (m, 1H, CHCH2O), 3.68 (dd, J = 10.1, 5.6 Hz, 1H, CH2O), 3.713.75 (m, 1H, CH2O), 4.33 (br s, 1H, CHO), 4.90 (s, 1H, olefinic), 5.03 (s, 1H, olefinic), 7.377.43 (m, 6H, aromatic), 7.66-7.72 (m, 4H, aromatic);
13
C NMR (100 MHz, CDCl3): δ = 9.7 (CH3CH), 19.2 (quarternary TBDPS), 19.4 (CH3C=C),
26.9 (3C, tBu), 37.3 (CHCH3), 68.0 (CH2OH), 76.3 (CHOH), 110.5 (olefinic CH2), 127.7,
129.7, 134.8, 135.6, 135.7 (aromatic), 145.7 (olefinic quarternary);
156
Experimental Section
HRMS (EI): calcd for C23H22O2Si [M+Na]+: 391.20631, found 391.20627.
(1S)-1-{(1R)-2[(tert-Butoxycarbonyl)amino]-1-methylethyl}-2-methylprop-2-enyl
propionate (147)
O
O
NHBoc
To a solution of alcohol 158 (130 mg, 0.57 mmol) in dry CH2Cl2 (5 mL) were added pyridine
(0.09 mL, 1.14 mmol) and n-propionyl chloride (0.07 mL, 0.79 mmol) at 0 oC. The reaction
mixture was warmed to room temperature and stirred for 12 h at room temperature. The
reaction mixture was diluted with CH2Cl2 (4 mL) and washed with brine, dried (Na2SO4),
filtered and concentrated in vacuo to give the crude product which was purified by flash
chromatography (1:9 ethylacetate/petroleum ether) providing the acylated product 147 (145
mg, 91%) as a colorless oil.
Rf = 0.40 (1:9 ethyl acetate/petroleum ether);
[α]20D = -15.6 (c 0.95, CH2Cl2);
IR (film): υmax = 3374, 2974, 2935, 1712, 1511, 1172 cm-1;
1
H NMR (400 MHz, CDCl3): δ = 0.84 (d, J = 6.8 Hz, 3H, CH3CH), 1.15 (t, J = 7.6 Hz, 3H,
CH2CH3), 1.42 (s, 9H, tBu), 1.69 (s, 3H, CH3C=C), 2.01-2.04 (m, 1H, CHCH3), 2.37 (q, J =
7.6 Hz, 2H, CH2CH3), 2.78-2.85 (m, 1H, CH2NH), 3.08-3.15 (m, 1H, CH2NH), 4.90 (br s, 3H,
NH, olefinic CH), 5.19 (br s, 1H, CHO);
13
C NMR (100 MHz, CDCl3): δ = 9.2 (CH2CH3), 11.8 (CH3CH), 19.3 (CH3C=C), 27.7
(CH2CH3), 28.4 (3C, tBu), 35.1 (CHCH3), 43.2 (CH2NH), 76.4 (CHOH), 79.2 (Boc
quarternary), 112.1 (CH2 olefinic), 141.9 (olefinic quarternary), 156.0 (Boc C=O), 174.0 (ester
C=O);
HRMS (EI): calcd for C15H27NO4 [M+Na]+: 308.18323, found 308.18339.
Experimental Section
157
(2R,3S)-3-{[tert-Butyl(dimethyl)silyl]oxy}-2,4-dimethylpent-4-en-1-ol (150)
OR
OH
R = TBS
To a solution of protected aldol product 151 (500 mg, 1.20 mmol) in THF (32 mL) was added
NaBH4 (225 mg, 6.00 mmol) in H2O (12 mL) at 0 oC. The reaction stirred for 7 h at room
temperature. The reaction mixture was treated with saturated aq. NH4Cl (10 mL) and stirred
for 1 h at room temperature. After separation of the layers, the aqueous layer extracted with
ethyl acetate (3 x 25 mL). The combined organic layers were washed with saturated aq.
NaHCO3 (20 mL), brine (20 mL), dried (Na2SO4), filtered and concentrated in vacuo to give
the crude product. The crude product was purified by flash chromatography (1:3 ethyl
acetate/petroleum ether) to afford the pure diol 150 (220 mg, 76%) as colorless oil.
Rf = 0.23 (1:3 ethyl acetate/petroleum ether);
1
H NMR (400 MHz, CDCl3): δ = 0.05, 0.01 (two s, 6H, Si(CH3)2), 0.85 (d, J = 7.1 Hz, 3H,
CH3CH), 0.89 (s, 9H, tBu), 1.70 (s, 3H, CH3C=C), 1.81-1.87 (m, 1H, CHCH2O), 3.47 (dd, J =
10.9, 5.3 Hz ,1H, CH2O), 3.56 (dd, J = 10.7, 7.0 Hz,1H, CH2O), 4.22 (d, J = 5.1 Hz, 1H,
CHOTBS), 4.87 (s, 1H, olefinic), 4.91 (s, 1H, olefinic);
13
C-NMR (100 MHz, CDCl3): δ = -4.7, -5.3 (Si(CH3)2), 11.9 (CH3CH), 18.2 (quarternary
TBS), 18.6 (CH3C=C), 25.8 (3C, tBu), 39.4 (CHCH3), 65.9 (CH2OH), 78.1 (CHOTBS), 112.0
(olefinic CH2), 146.4 (olefinic quarternary).
(2R,3S)-3-{[tert-Butyl(dimethyl)silyl]oxy}-2,4-dimethylpent-4-enyl 4-methoxybenzene
sulfonate (152)
OTBS
OTs
158
Experimental Section
To a solution of alcohol 150 (100 mg, 0.41 mmol) in pyridine (1 mL) was added tosyl chloride
(260 mg, 1.34 mmol) at 0 oC and the solution stirred for 4 h at 0 oC. Pre cooled water (1 mL)
was added and the reaction mixture stirred for 30 min at 0 oC. The reaction mixture was
diluted with diethyl ether (5 mL) and the layers were separated. The aqueous layer was
extracted with diethyl ether (2 x 3 mL) and the combined organic layers were washed with
brine (5 mL), dried (Na2SO4), filtered and evaporated to give the crude product which was
purified by flash chromatography (1:9 ethyl acetate/petroleum ether) providing the pure
tosylated product 152 (150 mg, 92%) as a colorless gel.
Rf = 0.40 (1:9 ethyl acetate/petroleum ether);
1
H NMR (400 MHz, CDCl3): δ = -0.06, -0.03 (two s, 6H, Si(CH3)2), 0.82 (d, J = 6.8 Hz, 3H,
CH3CH), 0.84 (s, 9H, tBu), 1.91 (s, 3H, CH3C=C), 1.85-1.94 (m, 1H, CHCH2O), 2.44 (3H,
methyl(toluene)), 3.78 (dd, J = 9.5, 6.4 Hz, 1H, CH2O), 3.91-3.95 (m, 2H, CH2O,CHOTBS),
4.87 (s, 1H, olefinic), 4.91 (s, 1H, olefinic), 7.33 (d, J = 8.1 Hz, 2H, aromatic), 7.77 (d, J = 8.3
Hz, 2H, aromatic);
13
C NMR (100 MHz, CDCl3): δ = -5.3, -4.6 (Si(CH3)2), 11.2 (CH3CH), 18.1 (CH3C=C), 21.6
(methyl(toluene)), 25.8 (3C, tBu), 36.7 (CHCH3), 72.3 (CH2OH), 75.7 (CHOTBS), 112.5
(olefinic CH2), 127.9, 129.8, 133.1, 144.7 (aromatic), 146.4 (olefinic quarternary);
(2S,3S)-3-{[tert-Butyl(dimethyl)silyl]oxy}-2,4-dimethylpent-4-enamide (154)
OR O
NH2
R = TBS
To a solution of acid 155 (400 mg, 1.54 mmol) in dry CHCl3 (10 mL) were added NH4HCO3
(360 mg, 4.50 mmol) and EEDQ (410 mg, 1.66 mg) at room temperature. The mixture was
stirred for 48 h at room temperature, then diluted with dichoromethane (15 mL). The reaction
mixture was washed with water (10 mL) and then with brine (10 mL). The dried (Na2SO4)
organic layer was filtered and concentrated in vacuo to give the crude product which was
Experimental Section
159
purified by flash chromatography (1:1 ethylacetate/petroleum ether) providing the pure amide
154 (300 mg, 75%) as a colorless oil.
Rf = 0.40 (1:1 ethylacetate/petroleum ether);
[α]20D = -4.5 (c 1.18 CH2Cl2);
IR (film): υmax = 3340, 3193, 2935, 2892, 1662, 1461, 1072 cm-1;
1
H NMR (400 MHz, CDCl3): δ = -0.03 (s, 3H, CH3TBS), 0.02 (s, 3H, CH3TBS), 0.86 (s, 9H,
tBu), 1.07 (d, J = 7.1 Hz, 3H, CH3CH), 1.66 (s, 3H, CH3C=C), 2.40-2.46 (m, 1H, CHCO),
4.21 (d, J = 5.8 Hz, 1H, CHO), 4.84 (s, 1H, olefinic), 4.91 (s, 1H, olefinic), 5.91, 6.13 (br s,
2H, amide);
13
C NMR (100 MHz, CDCl3): δ = -5.4, -4.8 (CH3TBS), 12.5 (CH3CH), 18.0 (CH3C=C), 18.1
(quarternary TBS), 25.8 (3C, tBu), 45.4 (CHCO), 77.8 (CHOTBS), 113.2 (CH2 olefinic),
144.7 (olefinic quarternary), 177.2 (CONH2);
HRMS (EI): calcd for C13H27NO2Si [M+Na]+: 280.17033, found 280.17021.
(2S,3S)-3-{[tert-Butyl(dimethyl)silyl]oxy}-2,4-dimethylpent-4-enoic acid (155)
OR O
OH
R = TBS
To a solution of the protected aldol product 151 (1.00 g, 2.4 mmol) in THF (25 mL) was added
H2O2 (1.2 mL of a 30 wt% solution, 9.6 mmol) at 0 oC through a syringe, followed by the
addition of LiOH·H2O (200 mg, 4.80 mmol) in water (12 mL). The solution was stirred at 0 oC
for 5 h. Subsequently, saturated Na2SO3 solution (10 mL) and saturated NaHCO3 solution (10
mL) were added at 0 oC. The whole mixture was partially concentrated in vacuo and diluted
with water (10 mL). The aqueous layer was extracted with dichloromethane (3 x 25 mL) to
recover the auxiliary. The aqueous layer was then acidified at 0 oC to pH 3 by using 1N HCl
and then extracted with ethyl acetate (4 x 25 mL). The combined ethyl acetate layers were
dried (MgSO4), filtered, and concentrated to furnish an oily residue. Due to presence of some
of the desired acid in the dichloromethane layer along with the chiral auxiliary, this mixture
160
Experimental Section
was purified by using flash chromatography (1:3 ethyl acetate/petroleum ether) giving the pure
acid 155 (480 mg, 77% from both dichloromethane and ethyl acetate layers) as a colorless oily
residue.
Rf = 0.35 (1:3 ethyl acetate/petroleum ether);
[α]20D = -11.4 (c 1.10 CH2Cl2);
IR (film): υmax = 3100, 2938, 2892, 2618, 1708, 1461, 1079 cm-1;
1
H NMR (400 MHz, CDCl3): δ = -0.01(s, 3H, CH3TBS), 0.02 (s, 3H, CH3TBS), 0.87 (s, 9H,
tBu), 1.11 (d, J = 7.1 Hz, 3H, CH3CH), 1.69 (s, 3H, CH3C=C), 2.59-2.65 (m, 1H, CHCO),
4.32 (d, J = 5.8 Hz, 1H, CHO), 4.86 (s, 1H, olefinic), 4.95 (s, 1H, olefinic), 11.42 (br s,
CO2H);
13
C NMR (100 MHz, CDCl3): δ = -5.4, -4.7 (CH3TBS), 11.3 (CH3CH), 17.7 (CH3C=C), 18.1
(quarternary TBS), 25.7 (3C, tBu), 44.3 (CHCO), 77.3 (CHOTBS), 113.2 (CH2 olefinic),
144.7 (olefinic quarternary), 180.8 (CO2H);
HRMS (EI): calcd for C13H26O3Si [M+Na]+: 281.15434, found 281.15424.
tert-butyl (2R,3S)-3-hydroxy-2,4-dimethylpent-4-enylcarbamate (158)
OH
NHBoc
To a solution of amide 154 (275 mg, 1.07 mmol) in dry THF (5 mL) was added LiAlH4 (1 M
solution in ether, 4.0 mL, 4.00 mmol) at 0 oC. The mixture was stirred for 0.5 h at 0 oC, then
gradually brought to room temperature by removing the ice bath. Thereafter, the mixture was
heated to reflux for 2 h. The mixture was cooled to room temperature and carefully worked up
by the dropwise and sequential addition of 0.2 mL of water, 0.2 mL of 15% aqueous NaOH
and an additional 0.5 mL of water. The reaction mixture was filtered through a bed of celite
and the celite bed washed thoroughly with ether (6 x 10 mL). The combined filtrates were
dried (MgSO4), and concentrated in vacuo to produce the crude amino alcohol which was used
for next step (Boc protection) without any further purification.
Experimental Section
161
To the crude aminol in dry CH2Cl2 (5 mL) were added NEt3 (0.27 mL, 2.00 mmol) and
Boc anhydride (270 mg, 1.25 mmol) at room temperature. After stirring for 12 h, the reaction
mixture was acidified up to pH 3 by using 5% aqueous KHSO4. The reaction mixture was
extracted with dichloromethane (3 x 5 mL). The combined organic layers were washed with
saturated aq. NaHCO3 (5 mL), brine (5 mL), dried (Na2SO4), filtered and concentrated in
vacuo to give the crude protected aminol which was purified by flash chromatography (1:3
ethyl acetate/petroleum ether) to provide the product 158 (145 mg, 59% two steps) as a
colorless oil.
Rf = 0.35 (1:3 ethyl acetate/petroleum ether);
[α]20D = -4.9 (c 1.00, CH2Cl2);
IR (film): υmax = 3363, 2973, 2931, 1689, 1523, 1072 cm-1;
1
H NMR (400 MHz, CDCl3): δ = 0.76 (d, J = 6.8 Hz, 3H, CH3CH), 1.42 (s, 9H, tBu), 1.67 (s,
3H, CH3C=C), 1.75-1.80 (m, 1H, CHCH3), 2.91-2.98 (m, 2H, CH2NH, OH), 3.24-3.31 (m,
1H, CH2NH), 4.02 (br s, 1H, CHOH), 4.90 (br s, 2H, NH, olefinic CH), 5.03 (s, 1H, olefinic
CH);
13
C-NMR (100 MHz, CDCl3): δ = 10.6 (CHCH3), 19.5 (CH3C=C), 28.4 (3C, tBu), 36.3
(CHCH3) 43.8 (CH2NH), 74.1 (CHOH), 79.1 (Boc quarternary), 110.5 (CH2 olefinic), 145.7
(olefinic quarternary), 157.1 (Boc C=O);
HRMS (EI): calcd for C12H23NO3 [M+Na]+: 252.15701, found 252.15697.
(3S,6R,9S,12R,16R)-6-(4-Hydroxybenzyl)-3-isopropyl-7,9,12,14,16-pentamethyl-1-oxa4,7,10-triazacycloheptadec-14-ene-2,5,8,11-tetrone (159)
O
HN
HO
N
O
O
N
H
O
O
162
Experimental Section
To a solution of linear depsipeptide 161 (40 mg, 0.05 mmol) in CH2Cl2 (0.5 mL) was added
TFA (0.08 mL, 0.99 mmol) at 0 oC. Stirring was continued for 2 h, at this time TLC showed
the complete consumption of reactant 161. The solvent was removed in vacuo and the residue
dried by azeotropic removal of H2O with toluene. The crude material was used for the next
step without further purification. To a solution of crude amine salt in dry DMF (50 mL) were
added DIEA (0.04 mL, 0.20 mmol), HOBt (20 mg, 0.15 mmol) and TBTU (48 mg, 0.15
mmol) successively at room temperature. The solution was stirred at room temperature for 18
h and then partitioned between ethyl acetate and water. The aqueous layer was extracted with
ethyl acetate (3 x 30 mL). The combined ethyl acetate layers were washed successively with
5% aqueous KHSO4, water, half saturated aq. NaHCO3, brine, dried (MgSO4), filtered and
concentrated in vacuo to give the crude product which was purified by flash chromatogaphy
(1:1 ethyl acetate/petroleum ether) providing the pure cyclic depsipeptide (12 mg, 39%) as a
colorless oil.
To the above TBS protected cyclic depsipeptide (12 mg, 0.02 mmol) in THF (0.2 mL)
was added TBAF containing 5% water (1 M solution in THF, 0.04 ml, 0.04 mmol) at 0 oC.
The solution was stirred for 3 h at 0 oC. The mixture was concentrated in vacuo and the crude
macrocycle purified by flash chromatography (7:3 ethyl acetate/petroleum ether) yielding the
pure cyclic depsipeptide 159 (8 mg, 80%) as a colorless oil.
Rf = 0.22 (7:3 ethyl acetate/petroleum ether);
[α]20D = +11.6 (c 0.70, CH2Cl2);
IR (film): υmax = 3361, 2974, 2930, 1735, 1712, 1511, 1172 cm-1;
1
H NMR (600 MHz, DMSO-d6): δ = 0.86 (CH3CHCH2O), 0.87 (Val Me), 0.93 (d, J = 6.7 Hz,
3H, Val Me), 0.95 (d, J = 6.6 Hz, 3H, Ala Me), 1.01 (d, J = 6.9 Hz, 3H, CH3CHCO), 1.56 (s,
3H, CH3C=C), 1.83 (d, J = 14.9 Hz, 1H, CH2hCHCO), 2.02 (p-sextett. J = 6.7 Hz, 1H, Val
CH), 2.25 (dd, J = 15.1, 11.1 Hz, 1H, CH2tCHCO), 2.36-2.43 (m, 1H, CHCO), 2.56-2.63 (m,
1H, CHCH2O), 2.66 (dd, J = 14.3, 8.1 Hz, 1H, Tyr CH2h), 2.79 (s, 3H, NMe), 3.00 (dd, J =
14.3, 6.4 Hz, 1H, Tyr CH2t), 3.70 (dd, J = 10.8, 2.5 Hz CH2hO), 4.04 (pt, J = 7.7 Hz, Val CH),
4.18 (dd J = 10.9, 5.4 Hz, 1H, CH2tO), 4.52 (qn, J = 6.9 Hz, 1H, Ala CH), 5.05 (d, J = 8.0 Hz,
olefinic), 5.24 (dd, J = 8.7, 6.5 Hz, 1H, Tyr CH), 6.61 (d, J = 8.5 Hz, 2H, TyrArHmeta), 6.98 (d,
Experimental Section
163
J = 8.5 Hz, 2H, TyrArHortho), 7.43 (d, J = 7.7 Hz, 1H, Val NH), 8.05 (d, J = 7.8 Hz, 1H, Ala
NH), 9.05 (s, Tyr-OH);
13
C NMR (600 MHz, DMSO-d6): δ = 16.9 (Ala CH3), 17.8 (CH3C=C), 18.8 (Val CH3), 19.5
(CH3CHCO), 29.1 (Val CH), 29.8 (NCH3), 31.3 (CHCH2O), 32.4 (Tyr CH2), 38.5 (CHCO),
40.6 (CH2CHCO), 44.0 (Ala CH), 56.3 (Tyr CH), 58.8 (Val CH), 68.1 (CH2O), 114.4 (TyrAr
meta), 125.2 (CH olefinic), 127.7 (CipsoAr), 129.5 (TyrArortho), 133.8 (olefinic quarternary),
155.3 (C-OH Ar), 169.8 (Tyr CO), 170.2 (Val CO), 171.5 (Ala CO), 174.1 (CH2CHCO);
HRMS (EI): calcd for C28H41N3O6Si [M+Na]+: 538.28876, found 538.28906.
(3S,6R,9S,12R,16R)-6-(4-Hydroxybenzyl)-3-isopropyl-7,9,12,14,16-pentamethyl-1,4,7,10tetraazacycloheptadec-14-ene-2,5,8,11-tetrone (160)
H
N
HN
HO
N
O
O
O
O
N
H
0.4 N LiOH.H2O (0.08 mL, 0.03 mmol) was added dropwise to a stirring solution of linear
tetrapeptide 181 (20 mg, 0027 mmol) in THF (0.5 mL). The reaction mixture was stirred for 2
h at room temperature before it was acidified up to pH 3 using 1 N HCl and extracted using
ethyl acetate (3 x 2 mL). The combined organic layers were dried (Na2SO4), filtered and
concentrated in vacuo to furnish the TBS deprotected free acid in quantitative yield. This acid
was used for the next step without further purification. To a solution of the above N-Boc
protected free acid (17 mg, 0.027 mmol) in CH2Cl2 (0.3 mL) was added TFA (0.02 mL, 0.99
mmol) at 0 oC. Stirring was continued for 2 h. The solvent was concentrated in vacuo and the
residue dried by azeotropic removal of H2O with toluene. The crude material was used for the
next reaction without any further purification. To a solution of crude amine salt in dry DMF
(20 mL) were added DIEA (0.01 mL, 0.08 mmol), HOBt (8 mg, 0.06 mmol) and TBTU (19
mg, 0.06 mmol) successively at room temperature. The solution was stirred at room
temperature for 18 h and then partitioned between ethyl acetate and water. The aqueous layer
164
Experimental Section
was extracted with ethyl acetate (3 x 30 mL). The combined ethyl acetate layers were washed
successively with 5% aqueous KHSO4, water, half saturated aq. NaHCO3, brine, dried
(MgSO4), filtered and concentrated in vacuo to give the crude product which was purified by
flash chromatography (ethyl acetate) to deliver the macrolactam 160 (6 mg, 45%) as a
colorless oil.
Rf = 0.55 (ethyl acetate);
[α]20D = -72.0 (c 0.40, CH2Cl2);
IR (film): υmax = 3361, 2974, 2930, 1735, 1712, 1511, 1172 cm-1;
1
H NMR (600 MHz, DMSO-d6): δ = 0.66 (dd, J = 3.7, 3.6 Hz, 6H, Val CH3), 0.84 (d, J = 7.0
Hz, 3H, CH3CHCH2NH), 0.98 (d, J = 6.9 Hz, 3H, CH3CHCO), 1.04 (d, J = 7.0 Hz, 3H, Ala
Me), 1.46 (s, 3H, CH3C=C), 1.76 (d, J = 15.9 Hz, 1H, CH2hCHCO), 2.20 (m, 2H, Val CH,
CH2tCHCO), 2.53 (m, 1H, CHCO), 2.54 (m, 1H, CHCH2NH), 2.83 (ddd, J = 12.9, 5.6, 2.6 Hz,
1H, CH2tNH), 2.91 (d, J = 8.2 Hz, 2H, Tyr CH2), 3.06 (s, 3H, NMe), 3.14 (ddd, J = 13.1, 9.1,
6.0 Hz, 1H, CH2hNH), 4.06 (dd, J = 9.6, 4.9 Hz, Val CH), 4.59 (qn, J = 6.9 Hz, 1H, Ala CH),
4.96 (d, J = 8.0 Hz, olefinic), 5.01 (t, J = 8.2 Hz, 1H, Tyr CH), 6.64 (d, J = 8.5 Hz, 2H,
TyrArHmeta), 7.03 (d, J = 8.5 Hz, 2H, TyrArHortho), 7.43 (t, J = 5.8 Hz, 1H, CH2NH), 7.87 (d, J
= 9.7 Hz, 1H, Val NH), 8.00 (d, J = 7.0 Hz, 1H, Ala NH), 9.10 (s, Tyr-OH);
13
C NMR (600 MHz, DMSO-d6): δ = 16.7 (Val CH3), 16.9 (Ala CH3), 17.8 (CH3C=C), 18.8
(CH3CHCH2NH), 18.9 (Val CH3), 19.5 (CH3CHCO), 28.1 (Val CH), 30.7 (NCH3), 31.6
(CHCH2NH), 32.9 (Tyr CH2), 36.7 (CHCO), 41.1 (CH2CHCO), 44.3 (Ala CH), 45.7
(CH2NH), 56.7 (Val CH), 57.6 (Tyr CH), 114.6 (TyrArmeta), 126.7 (CH olefinic), 126.8 (C
ipsoAr),
129.2 (TyrArortho), 133.6 (olefinic quarternary), 155.5 (C-OH Ar), 170.4 (Val CO),
170.9 (Tyr CO), 174.4 (CH2CHCO), 174.8 (Ala CO);
HRMS (EI): calcd for C28H42N4O5Si [M+Na]+: 537.30474, found 537.30438.
Experimental Section
165
(2S,3E,6S)-7-tert-Butoxy-2,4,6-trimethyl-7-oxohept-3-enyl N-(tert-butoxycarbonyl)-Lalanyl-O-[tert-butyl(dimethyl)silyl]-N-methyl-D-tyrosyl-L-valinate (161)
O
HN
TBSO
N
O
O
O
O
t
NHBoc
O Bu
To a solution of acid 162 (70 mg, 0.15 mmol), amine 163 (50 mg, 0.15 mmol) in dry DMF
(1.5 mL) were added DIEA (0.07 mL, 0.44 mmol), HOBt (20 mg, 0.15 mmol) and TBTU (47
mg, 0.15 mmol) at room temperature. The reaction mixture was stirred for 5 h at room
temperature, before it was treated with water (2 mL) and stirred for further 5 min and extracted
with ethyl acetate (3 x 4 mL). The combined ethyl acetate layers were washed with 1 N HCl (3
mL), saturated aq. NaHCO3 (3 mL), brine, dried (Na2SO4), filtered and concentrated in vacuo
to give the crude product which was purified by flash chromatography (15:85 ethyl
acetate/petroleum ether) providing the pure linear depsipeptide 161 (60 mg, 51%) as a colorles
oil.
Rf = 0.37 (15:85 ethyl acetate/petroleum ether);
[α]20D = +28.0 (c 0.50, CH2Cl2);
IR (film): υmax = 3361, 2974, 2930, 1735, 1712, 1511, 1172 cm-1;
1
H NMR (400 MHz, CDCl3): δ = 0.12 (s, 6H, CH3TBS), 0.85 (d, J = 6.1 Hz, 3H, Val CH3),
0.87 (d, J = 6.1 Hz, 3H, Val CH3), 0.89 (d, J = 6.8 Hz, 3H, CH3CHCH2O), 0.93-0.95 (m, 12H,
tBu(TBS), CH3CHCO), 1.02 (d, J = 6.8 Hz, 3H, Ala CH3), 1.38,1.40 (two s, 18H, tBu, Boc
tBu), 1.60 (s, 3H, CH3C=C), 1.91-1.97 (m, 1H, Val CH), 2.10-2.18 (m, 1H, CH2CHCO), 2.282.34 (m, 1H, CH2CHCO), 2.42-2.48 (m, 1H, CHCO), 2.70-2.75 (m, 1H, CHCH2O), 2.84-2.88
(m, 1H, Tyr CH2), 2.91 (s, 3H, NCH3), 3.29 (dd, J = 14.9, 5.8 Hz, 1H, Tyr CH2), 3.84-3.92 (m,
1H, CH2O), 4.39-4.45 (m, 1H, Ala CH), 4.92 (d, J = 8.8 Hz, CH olefinic), 5.25 (d, J = 7.1 Hz,
Val CH), 5.28 (br s, 1H, Ala NH), 5.49 (dd, J = 10.4, 5.8 Hz, 1H, Tyr CH), 6.59 (d, J = 8.8
Hz, 1H, Val NH), 6.69 (d, J = 8.1 Hz, 2H, aromatic), 7.01 (d, J = 8.3 Hz, 2H, aromatic);
166
13
Experimental Section
C NMR (100 MHz, CDCl3): δ = -4.5 ( 2C, CH3TBS), 16.0 (CH3C=C), 16.8 (CH3CHCO),
17.6 (Val CH3), 17.7 (Val CH3, CH3CH2CHO), 18.2 (quarternary C, tBu (TBS)), 19.1 (Ala
CH3), 25.6 (3C, tBu (TBS)), 28.1, 28.3 (6C, tBu, Boc tBu), 30.6 (CHCH2O), 30.8 (Val CH),
31.8 (NCH3), 34.3 (Tyr CH2), 38.6 (CHCO), 43.9 (CH2CHO), 46.6 (Ala CH), 57.2 (Val CH),
57.4 (Tyr CH), 69.5 (CH2O), 79.6, 79.9 (2C, quarternary C (Boc, tBu)), 120.0, 128.2, 129.4
(aromatic), 129.7 (CH olefinic), 134.4 (olefinic quarternary), 154.4 (Boc C=O), 155.3
(phenolic), 170.1 (Tyr CO), 171.7 (Val CO), 174.5 (Ala CO), 176.4 (CO2tBu);
HRMS (EI): calcd for C43H73N3O9Si [M+Na]+: 826.50083, found 826.50078.
N-(tert-Butoxycarbonyl)-L-alanyl-O-[tert-butyl(dimethyl)silyl]-N-methyl-D-tyrosine (162)
OH
TBSO
N
160
O
O
N
H
Boc
To a solution of dipeptide 175 (400 mg, 0.70 mmol) in ethanol (5 mL) was added 10% Pd/C
(80 mg). The reaction mixture was connected to a hydrogenation machine (Parr apparatus) and
shaked for 16 h in a hydrogen atmosphere at around 2 atm (30 psi) pressure at room
temperature. The reaction mixture was filtered through a bed of celite and the celite bed was
washed with ethyl acetate (2 x 5 mL). The filtrate was concentrated in vacuo to afford the
crude acid 162 which was used for the further reaction without purification.
1
H NMR (400 MHz, CDCl3): δ = 0.13 (s, 6H, CH3TBS), 0.86 (d, J = 7.6 Hz, 3H, Ala CH3),
0.94 (s, 9H, tBu(TBS)), 1.40, (s, 9H, Boc tBu), 2.85 (s, 3H, NCH3), 2.93-3.03 (m, 1H, Tyr
CH2), 3.34 (dd, J = 14.7, 4.3 Hz, 1H, TyrCH2), 4.47- 4.53 (m, 1H, Ala CH), 5.28 (dd, J = 11.2,
3.9 Hz, 1H, TyrCH), 5.56 (d, J = 8.1 Hz, 1H, Ala NH), 6.72 (d, J = 8.3 Hz, 2H, aromatic) 7.00
(d, J = 8.3, 2H, aromatic);
13
C NMR (100 MHz, CDCl3): δ = -4.6 ( 2C, CH3TBS), 18.2 (Ala CH3), 18.2 (quarternary C,
tBu (TBS)), 25.6 (3C, tBu (TBS)), 28.3 (Boc tBu), 32.6 (NCH3), 33.7 (Tyr CH2), 46.5 (Ala
Experimental Section
167
CH), 52.0 (OCH3), 58.4 (Tyr CH), 79.8 (quarternary C Boc), 120.2, 129.1, 129.7 (aromatic),
154.5 (Boc C=O), 155.3 (phenolic), 173.6 (Ala CO), 174.2 (Tyr CO).
tert-Butyl (2S,4E,6S)-2,4,6-trimethyl-7-(L-valyloxy)hept-4-enoate (163)
O
O
H2N
O
OtBu
Et2NH (7 mL) was added to a pre-cooled solution (0 oC) of F-moc protected valine ester 171
(150 mg, 0.27 mmol) in dry THF (7 mL). The reaction mixture was stirred for 15 min at 0 oC
and then at room temperature for 3 h. The solution was concentrated in vacuo and the resulting
oil was purified by flash chromatography (5:95 methanol/dichloromethane) to deliver the pure
amine 163 (80 mg, 88%) as a pale yellow oil.
Rf = 0.25 (5:95 methanol/dichloromethane);
IR (film): υmax = 3351, 2969, 2904, 1724, 1677, 1454, 1369, 1153 cm-1;
1
H NMR (400 MHz, CDCl3): δ = 0.88 (d, J = 6.8 Hz, 3H, Val CH3), 0.96 (d, J = 6.8 Hz, 6H,
Val CH3, CH3CH2O), 1.03 (d, J = 7.1 Hz, 3H, CH3CHCO), 1.41 (s, 9H, tBu), 1.62 (s, 3H,
CH3C=CH), 1.93-2.03 (m, 2H, CH2CHCO, Val CH), 2.13-2.20 (m, 1H, CHNH), 2.32 (dd, J =
13.3, 8.2 Hz, 1H, CH2CHCO), 2.43-2.51 (m, 1H, CHCH2O), 2.71-2.79 (m, 1H, CHCO), 3.883.98 (m, 2H, ValCH, CH2O), 4.11 (dd, J = 10.2, 8.2 Hz, 1H, CH2O), 4.22 (t, J = 7.0 Hz, 1H,
CHFmoc), 4.30 (dd, J = 9.0, 4.7 Hz, 1H, CH2Fmoc), 4.34-4.43 (m, 1H, CH2Fmoc), 4.94 (d, J
= 9.1 Hz, 1H, olefinic), 5.34 (d, J = 9.1 Hz, 1H, NH), 7.30 (t, J = 7.3 Hz, 2H, aromatic Fmoc),
7.39 (t, J = 7.3 Hz, 2H, aromatic Fmoc), 7.60 (d, J = 5.6 Hz, 2H, aromatic Fmoc), 7.75 (d, J =
7.7 Hz, 2H, aromatic Fmoc);
13
C NMR (100 MHz, CDCl3): δ = 16.0 (CH3C=CH), 16.8 (CH3CH2O), 17.5 (2C, Val CH3),
18.9 (CH3CHCO), 28.0 (3C, tBu), 31.4 (CHCH2O), 32.0 (Val CH), 38.6 (CHCO), 43.8
(CH2C=CH), 47.1 (Fmoc CH), 59.0 (CHNH), 67.0 (Fmoc CH2), 67.5 (CH2O), 79.8 (Boc
quarternary), 119.9, 125.1, 127.0, 127.7 (Fmoc aromatic), 128.1 (CH olefinic), 134.5 (C
168
Experimental Section
olefinic), 141.3, 143.7, 143.9 (Fmoc aromatic), 156.2 (NHCO), 172.1 (Val CO), 175.7
(CO2tBu);
HRMS (EI): calcd for C19H35NO4 [M+H]+: 342.26389, found 342.26393.
tert-Butyl (2S,4E,6S)-7-{[tert-Butyl(diphenyl)silyl]oxy}-2,4,6-trimethylhept-4-enoate (169)
ButO2C
OTBDPS
To a stirring solution of hydroxy acid 96 (300 mg, 0.71 mmol), DMAP (1.23 g, 10.61 mmol),
Et3N (1.00 mL, 7.10 mmol), and tBuOH (0.40 mL, 0.36 mmol) in dry toluene (70 mL) was
added 2,4,6-trichlorobenzoyl chloride (1.1 mL, 7.10 mmol) at -78 oC. After stirring for 30 min
at -78 oC, the reaction mixture was brought to room temperature by removing the cooling bath,
and then stirred additionally for 12 h at room temperature. The reaction mixture was treated
with saturated aqueous NaHCO3 (25 mL). After separation of the layers, the aqueous layer
was extracted with ethy lacetate (3 x 15 mL). The combined organic layers were washed with
brine (25 mL), dried (MgSO4), filtered, and concentrated in vacuo to afford the crude product
which was purified by flash chromatography (5:95 ethyl acetate/petroleum ether) giving the
pure product 169 (330 mg, 96%) as a colorless oil.
Rf = 0.22 (5:95 ethyl acetate/petroleum ether);
[α]20D = + 7.7 (c 0.71, CH2Cl2);
IR (film): υmax =3066, 2962, 2931, 2861, 1727, 1461, 1369, 1153, 1110 cm-1;
1
H NMR (400 MHz, CDCl3): δ = 0.97 (d, J = 6.8 Hz, 3H, CH3CH2O), 1.03 (d, J = 8.1 Hz, 3H,
CH3CO), 1.04 (s, 9H, tBuSi), 1.41 (s, 9H, tBu), 1.54 (s, 3H, CH3C=C), 1.95 (dd, J = 13.6, 7.3
Hz, 1H, CH2CO), 2.30 (dd, J = 13.4, 7.3 Hz, 1H, CH2CO), 2.41-2.50 (m, 1H, CHCO), 2.542.61 (m, 1H, CHCH2O), 3.39 (dd, J = 16.2, 6.8 Hz, 1H, CH2O), 3.45-3.49 (m, 1H, CH2O),
4.95 (d, J = 9.1 Hz, 1H, olefinic), 7.35-7.41 (m, 6H, aromatic), 7.66 (d, J = 6.8, 4H, aromatic);
13
C NMR (100 MHz, CDCl3): δ = 15.9 (CH3C=CH), 16.9 (CH3CH2O), 17.4 (CH3 CHCO),
19.2 (SiC(CH3)3), 26.8 (3C, SiC(CH3)3), 28.1 (3C, tBu), 35.4 (CHCH2O), 38.7 (CHCO), 44.1
Experimental Section
169
(CH2C=CH), 68.6 (CH2O), 79.7 (Boc quarternary), 127.5, 129.5 (aromatic), 129.8 (CH
olefinic), 132.8 (aromatic), 133.9 (olefinic), 135.6 (aromatic), 175.9 (CO2tBu);
HRMS (EI): calcd for C30H44O3Si [M+Na]+: 503.29519, found 503.29502.
tert-Butyl (2S,4E,6S)-7-hydroxy-2,4,6-trimethylhept-4-enoate (170)
ButO2C
OH
To a solution of protected hydroxy acid 169 (300 mg, 0.63 mmol) in THF (5 mL) was added
TBAF (1 M solution in THF containing 5% H2O, 0.75 mL, 0.75 mmol) at 0 oC. The reaction
mixture was stirred until the TLC showed the complete consumption of reactant (4-5 h). The
reaction mixture was concentrated in vacuo and purified by flash chromatography (1:3 ethyl
acetate/petroleum ether) to give the pure alcohol 170 (135 mg, 90%) as a colorless oil.
Rf = 0.35 (1:3 ethyl acetate/petroleum ether);
[α]20D = -15.7 (c 0.77, CH2Cl2);
IR (film) υmax = 3384, 2973, 2930, 2872, 1729, 1457, 1367, 1151 cm-1;
1
H NMR (400 MHz, CDCl3): δ = 0.90 (d, J = 6.6 Hz, 3H, CH3CH2O), 1.06 (d, J = 7.1 Hz, 3H,
CH3CHCO), 1.41 (s, 9H, tBu), 1.65 (s, 3H, CH3C=CH), 2.05-2.07 (m, 1H, CH2CHCO), 2.32
(dd, J = 13.3, 8.2 Hz, 1H, CH2CHCO), 2.46-2.55 (m, 1H, CHCO), 2.56-2.65 (m, 1H,
CHCH2O), 3.29 (dd, J = 10.2, 8.2 Hz, 1H, CH2O), 3.44 (dd, J = 10.4, 5.8 Hz, 1H, CH2O), 4.90
(d, J = 9.4, 1H, olefinic);
13
C NMR (100 MHz, CDCl3): δ = 16.6 (CH3C=CH), 16.9 (CH3CH2O), 17.1 (CH3CHCO),
28.1 (3C, tBu), 35.5 (CHCH2O), 39.0 (CHCO), 43.8 (CH2C=CH), 67.8 (CH2O), 80.0 (Boc
quarternary), 129.1 (CH olefinic), 135.3 (C olefinic), 175.8 (CO2tBu);
HRMS (EI): calcd for C34H45NO6 [M+Na]+: 265.17742, found 265.17750.
170
Experimental Section
tert-Butyl (2S,4E,6S)-7-({N-[(9H-fluoren-9-ylmethoxy) carbonyl]–L-valyl}oxy)-2,4,6trimethylhept-4-enoate (171)
O
tBuO2C
O
NHFmoc
To a solution of hydroxy acid derivative 170 (100 mg, 0.41 mmol), Fmoc-L-Valine (140 mg,
0.41 mmol) and DMAP (25 mg, 0.20 mmol), in dry CH2Cl2 (4 mL) was added a solution of
DCC (110 mg, 0.53 mmol) in dry CH2Cl2 (0.6 mL) dropwise at 0 oC. The reaction mixture
was stirred for 0.5 h at 0 oC, then stirred for 10 h at room temperature. The reaction mixture
was diluted with diethyl ether (10 mL) and filtered to remove the cyclohexyl urea and the
precipitate washed twice with diethyl ether (5 mL). The filtrate was concentrated to give the
crude product which was purified by flash chromatography (1:4 ethyl acetate/petroleum ether)
providing the pure product 171 (205 mg, 88%) as a colorless gel.
Rf = 0.30 (1:4 ethyl acetate/petroleum ether);
[α]20D = -5.9 (c 0.87, CH2Cl2);
IR (film): υmax = 3351, 2969, 2904, 1724, 1677, 1454, 1369, 1153 cm-1;
1
H NMR (400 MHz, CDCl3): δ = 0.90 (d, J = 6.8 Hz, 3H, CH3CH2O), 0.97 (d, J = 6.3 Hz, 6H,
Val CH3), 1.03 (d, J = 6.8 Hz, 3H, CH3CHCO),1.41 (s, 9H, tBu), 1.62 (s, 3H, CH3C=CH),
1.96 (dd, J = 13.6, 7.3 Hz, 1H, CH2CHCO), 2.13-2.20 (m, 1H, CHNH), 2.32 (dd, J = 13.3, 8.2
Hz, 1H, CH2CHCO), 2.43-2.51 (m, 1H, CHCH2O), 2.71-2.79 (m, 1H, CHCO), 3.88-3.98 (m,
2H, ValCH, CH2O), 4.11 (dd, J = 10.2, 8.2 Hz, 1H, CH2O), 4.22 (t, J = 7.0 Hz, 1H, CHFmoc),
4.30 (dd, J = 9.0, 4.7 Hz, 1H, CH2Fmoc), 4.34-4.43 (m, 1H, CH2Fmoc), 4.94 (d, J = 9.1 Hz,
1H, olefinic), 5.34 (d, J = 9.1 Hz, 1H, NH), 7.30 (t, J = 7.3 Hz, 2H, aromatic Fmoc), 7.39 (t, J
= 7.3 Hz, 2H, aromatic Fmoc), 7.60 (d, J = 5.6 Hz, 2H, aromatic Fmoc), 7.75 (d, J = 7.7 Hz,
2H, aromatic Fmoc);
13
C NMR (100 MHz, CDCl3): δ = 16.0 (CH3C=CH), 16.8 (CH3CH2O), 17.5 (2C, Val CH3),
18.9 (CH3CHCO), 28.0 (3C, tBu), 31.4 (CHCH2O), 32.0 (Val CH), 38.6 (CHCO), 43.8
(CH2C=CH), 47.1 (Fmoc CH), 59.0 (CHNH), 67.0 (Fmoc CH2), 67.5 (CH2O), 79.8 (Boc
quarternary), 119.9, 125.1, 127.0, 127.7 (Fmoc aromatic), 128.1 (CH olefinic), 134.5 (C
Experimental Section
171
olefinic), 141.3, 143.7, 143.9 (Fmoc aromatic), 156.2 (NHCO), 172.1 (Val CO), 175.7
(CO2tBu);
HRMS (EI): calcd for C34H45NO6 [M+Na]+: 586.31391, found 586.31401;
Benzyl N-(tert-butoxycarbonyl)-L-alanyl-O-[tert-butyl(dimethyl)silyl]-N-methyl-Dtyrosinate (175)
OBn
TBSO
N
O
O
N
H
Boc
To a solution of D-tyrosine benzylester derivative 174 (650 mg, 1.30 mmol) in CH2Cl2 (10
mL) was added CF3COOH (1.0 mL, 13.0 mmol), and the mixture was stirred at room
temperature for 1 h. The solvent was removed in vacuo, and the residue was dried by
azeotropic removal of H2O with toluene. The crude material was subjected to the next reaction
without further purification. To a stirred solution of crude amine, N-Boc-L-alanine (240 mg,
1.30 mmol), and PyBroP (635 mg, 1.30 mmol) in CH2Cl2 (5 mL) at 0 oC was added iPr2NEt
(0.8 mL, 4.70 mmol) and then the mixture was allowed to stir for 3 h at room temperature. The
solvent was removed in vacuo and the residue purified by flash chromatography (1:3 ethyl
acetate/petroleum ether) to give the dipeptide 175 (403 mg, 55%) as a colorless gel.
Rf = 0.45 (1:3 ethyl acetate/petroleum ether)
1
H NMR (400 MHz, CDCl3): δ = 0.13 (s, 6H, CH3TBS), 0.84 (d, J = 6.8 Hz, 3H, Ala CH3),
0.94 (s, 9H, tBu(TBS)), 1.41 (s, 9H, Boc tBu), 2.80 (s, 3H, NCH3), 2.95 (dd, J = 14.5, 11.8
Hz, 1H, TyrCH2), 3.33 (dd, J = 14.8, 4.9 Hz, 1H, TyrCH2), 4.43- 4.50 (m, 1H, Ala CH), 5.125.20 (m, 2H, CH2Ph), 5.26-5.30 (m, 1H, TyrCH), 5.44 (d, J = 7.8 Hz, 1H, Ala NH), 6.71 (d, J
= 8.6 Hz, 2H, aromatic), 6.99 (d, J = 8.6 Hz, 2H, aromatic), 7.31-7.34 (5H, aromatic CO2Bn);
13
C NMR (100 MHz, CDCl3): δ = -4.6 ( 2C, CH3TBS), 18.2 (Ala CH3), 18.2 (quarternary C,
tBu (TBS)), 25.6 (3C, tBu (TBS)), 28.3 (Boc tBu), 32.6 (NCH3), 33.7 (Tyr CH2), 46.5 (Ala
172
Experimental Section
CH), 52.0 (OCH3), 58.4 (Tyr CH), 79.8 (quarternary C Boc), 120.2, 129.1, 129.7 (aromatic),
154.5 (Boc C=O), 155.3 (phenolic), 173.6 (Ala CO), 174.2 (Tyr CO).
Methyl N-{(2S,4E,6S)-7-[(tert-butoxycarbonyl)amino]-2,4,6-trimethylhept-4-enoyl}-Lalanyl-O-[tert-butyl(dimethyl)silyl]-N-methyl-D-tyrosyl-L-valinate (181)
HN
TBSO
N
CO2Me
O
O
NHBoc
NH
O
To solution of tripeptide 182 (30 mg, 0.05 mmol) in CH2Cl2 (0.5 mL) was added TFA (0.03
mL, 0.5 mmol) at 0 oC. The resulting mixture was stirred for 1 h at 0 oC. The solvent was
removed in vacuo and the residue was dried by azeotropic removal of H2O with toluene. The
crude material was used for the next reaction without further purification. To a solution of
crude amine salt and amino acid 97 (15 mg, 0.05 mmol) in dry DMF (1 mL) were added DIEA
(0.02 mL, 0.25 mmol), HOBt (7 mg, 0.05 mmol) and TBTU (16 mg, 0.05 mmol) successively.
The reaction mixture was stirred for 2 h before it was diluted with water (2 mL), stirred for 5
min, and extracted with ethyl acetate (3 x 4 mL). The combined organic layers were washed
with 1 N HCl (2 mL), saturated aq. NaHCO3 (2 mL), brine (2 mL), dried (Na2SO4), filtered
and concentrated in vacuo to give the crude product which was purified by flash
chromatography (1:1 ethyl acetate/petroleum ether) producing the pure tetrapeptide (20 mg,
53%) as a colorless gel.
Rf = 0.52 (1:1 ethyl acetate/petroleum ether);
[α]20D = +16.6 (c 0.84, CH2Cl2);
IR (film): υmax = 3361, 2974, 2930, 1735, 1712, 1511, 1172 cm-1;
1
H NMR (400 MHz, CDCl3): δ = 0.13 (s, 6H, CH3TBS), 0.87 (d, J = 6.8 Hz, 3H, Val CH3),
0.88 (d, J = 6.8 Hz, 3H, Val CH3), 0.89 (d, J = 7.6 Hz, 3H, CH3CHCH2O), 0.91-0.94 (m, 12H,
Experimental Section
173
tBu (TBS), CH3CHCO), 1.04 (d, J = 6.8 Hz, 3H, Ala CH3), 1.41 (s, 9H, Boc tBu), 1.56 (s, 3H,
CH3C=C), 1.97-2.03 (m, 1H, Val CH), 2.13-2.21 (m, 1H, CHCH2NH), 2.27 (dd, J = 13.8, 6.7
Hz, 1H, CH2CHCO), 2.33-2.40 (m, 1H, CH2CHCO), 2.53-2.56 (m, 1H, CHCO), 2.75-2.81 (m,
1H, CH2NH), 2.85-2.89 (m, 1H, CH2NH), 2.93 (s, 3H, NCH3), 3.08-3.12 (m, 1H, Tyr CH2),
3.29 (dd, J = 14.9, 6.1 Hz, 1H, Tyr CH2), 3.69 (s, 3H, OCH3), 4.41 (dd, J = 8.5, 5.8 Hz, 1H,
Val CH), 4.61-4.68 (m, 1H, Ala CH), 4.75 (br s, 1H, NHBoc), 4.89 (d, J = 9.1 Hz, CH
olefinic), 5.48 (dd, J = 10.6, 6.1 Hz, 1H, Tyr CH), 6.37 (d, J = 5.8 Hz, 1H, Val NH), 6.70 (d, J
= 8.3 Hz, 2H, aromatic), 7.02 (d, J = 8.3 Hz, 2H, aromatic);
13
C NMR (100 MHz, CDCl3): δ = -4.5 (2C, CH3TBS), 16.4 (CH3C=C), 16.9 (CH3CHCO),
17.4 (Ala CH3), 17.9 (Val CH3), 18.2 (quarternary C, tBu (TBS)), 18.2 (Val CH3), 19.0
(CH3CHCH2NH), 25.6 (3C, tBu (TBS), 28.4 (3C, Boc tBu), 30.4 (NCH3), 30.8 (CHCH2NH),
32.8 (Val CH), 33.0 (Tyr CH2), 39.2 (CHCO) 43.8 (CH2CHCO), 45.5 (CH2NH), 46.5 (Ala
CH), 52.1 (OCH3), 57.1 (Tyr CH)), 57.7 (Val CH), 77.9 (quarternary C Boc), 120.1, 128.2,
129.3 (aromatic), 129.7 (CH olefinic), 130.5 (olefinic quarternary), 154.1 (phenolic), 156.1
(Boc C=O), 170.1 (Ala CO), 172.3 (Tyr CO), 174.3 (Val CO), 175.7 (CO2Me);
HRMS (EI): calcd for C40H68N4O8Si [M+Na]+: 783.46986, found 783.47035.
Methyl N-(tert-butoxycarbonyl)-L-alanyl-O-[tert-butyl(dimethyl)silyl]-N-methyl-Dtyrosyl-L-valinate (182)
HN
TBSO
N
CO2Me
O
O
NHBoc
To a solution of dipeptide acid 162 (200 mg, 0.42 mmol), and L-valine methyl ester
hydrochloride (70 mg, 0.42 mmol) in dry DMF (4 mL) were added DIEA (0.18 mL, 1.05
mmol), HOBt (58 mg, 0.42 mmol) and TBTU (135 mg, 0.42 mmol) at room temperature. The
resulting reaction mixture was stirred for 3 h at room temperature. The reaction mixture was
treated with water (5 mL), stirred for further 5 min, and extracted with ethyl acetate (3 x 10
174
Experimental Section
mL). The combined ethyl acetate layers were washed with 1 N HCl (5 mL), saturated aq.
NaHCO3 (5 mL), brine (5 mL), dried (Na2SO4), filtered, and concentrated in vacuo to furnish
the crude product which was purified by flash chromatography (1:3 ethyl acetate/petroleum
ether) yielding the pure tripeptide 182 (177 mg, 71%) as a colorless gel.
Rf = 0.44 (1:3 ethyl acetate/petroleum ether);
[α]20D = +45.4 (c 1.01, CH2Cl2);
IR (film): υmax = 3336, 2962, 2930, 1739, 1685, 1511, 1172, 1052 cm-1;
1
H NMR (400 MHz, CDCl3): δ = 0.11 (s, 6H, CH3TBS), 0.85 (d, J = 6.1 Hz, 3H, Val CH3),
0.87 (d, J = 6.1 Hz, 3H, Val CH3), 0.89 (d, J = 7.6 Hz, 3H, Ala CH3), 0.92 (s, 9H, tBu(TBS)),
1.37 (s, 9H, Boc tBu), 2.10-2.18 (m, 1H, Val CH), 2.84-2.87 (m, 1H, Tyr CH2), 2.91 (s, 3H,
NCH3), 3.28 (dd, J = 14.8, 5.4 Hz, 1H, Tyr CH2), 3.68 (s, 3H, OCH3), 4.39-4.42 (m, 2H, Ala
CH, Val CH), 5.26 (d, J = 6.3 Hz, 1H, Ala NH), 5.49 (dd, J = 9.9, 5.9 Hz, 1H, Tyr CH), 6.61
(d, J = 8.3 Hz, 1H, Val NH), 6.69 (d, J = 7.8 Hz, 2H, aromatic), 7.00 (d, J = 7.6 Hz, 2H,
aromatic);
13
C NMR (100 MHz, CDCl3): δ = -4.6 (2C, CH3TBS), 17.5 (Val CH3), 17.8 (Val CH3), 18.1
(quarternary C, tBu (TBS)), 19.0 (Ala CH3), 25.6 (3C, tBu (TBS)), 28.2 (Boc tBu), 30.4
(NCH3), 30.8 (Val CH), 32.7 (Tyr CH2), 46.6 (Ala CH), 52.0 (OCH3), 57.1 (Val CH), 57.5
(Tyr CH), 79.6 (quarternary C Boc), 120.0, 129.3, 129.7 (aromatic), 154.4 (Boc C=O), 155.3
(phenolic), 170.2 (Tyr CO), 172.0 (Ala CO), 174.8 (Val CO);
HRMS (EI): calcd for C30H51N3O7Si [M+Na]+: 616.33885, found 616.33956.
N-(tert-Butoxycarbonyl)-L-alanyl-N-{(1R)-3-methoxy-3-oxo-1-[4-(1,1,2,2tetramethylpropoxy)phenyl]propyl}-N-methyl-D-tryptophanamide (184)
Experimental Section
175
OTBS
O
H
H
N
Me
OMe
N
N
O
O
H
N
Boc
To solution of β-D tyrosine derivative 186 (90 mg, 0.23 mmol) in CH2Cl2 (2.0 mL) was added
TFA (0.18 mL, 2.3 mmol) at 0 oC. The reaction mixture was stirred for 1 h at 0 oC. The
solvent was removed in vacuo and residue dried by azeotropic removal of H2O with toluene.
The crude material was used for the next reaction without further purification. To a solution of
the crude amine salt, and dipeptide acid 192 (90 mg, 0.23 mmol) in dry DMF (2 mL) were
added iPr2NEt (0.09 mL, 0.56 mmol), HOBt (32 mg, 0.23 mmol), and TBTU (74 mg, 0.23
mmol) successively. The resulting mixture was stirred for 2 h. The reaction mixture was
diluted with water (2 mL), stirred for 5 min and extracted with ethyl acetate (3 x 4 mL). The
combined organic layers were washed with 1 N HCl (2 mL), saturated aq. NaHCO3 (2 mL),
brine (2 mL), dried (Na2SO4), filtered and concentrated in vacuo. The crude product was
purified by flash chromatography (1:1 ethyl acetate/petroleum ether) to furnish the pure
tripeptide 184 (100 mg, 70%) as a colorless gel.
Rf = 0.44 (1:1 ethyl acetate/petroleum ether);
[α]20D = +22.0 (c 0.423, CH2Cl2);
IR (film): υmax = 3335, 2935, 2850, 1730, 1680, 1515, 1260, 1170 cm-1;
1
H NMR (400 MHz, CDCl3): δ = 0.16 (s, 6H, CH3TBS), 0.90 (d, J = 6.8 Hz, 3H, Ala CH3),
0.95 (s, 9H, tBu(TBS)), 1.39 (s, 9H, Boc tBu), 2.75 (dd, J = 15.5, 5.4 Hz, 1H, β-Tyr CH2),
2.82-2.88 (m, 1H, β-Tyr CH2), 2.91 (s, 3H, NCH3), 3.21 (dd, J = 15.6, 10.5 Hz, 1H, Trp CH2),
3.42 (dd, J = 15.9, 5.3 Hz, 1H, Trp CH2), 3.59 (s, 3H, OCH3), 4.39 (qn, J = 6.8 Hz, 1H, Ala
CH), 5.34-5.40 (m, 1H, β-Tyr CH), 5.61 (dd, J = 10.1, 5.6 Hz, Trp CH), 6.72 (d, J = 8.3 Hz,
2H, β-Tyr aromatic), 6.93 (s, 1H, Trp aromatic), 7.00 (d, J = 8.3 Hz, 1H, β-Tyr NH), 7.07 (d, J
= 7.8 Hz, 2H, Tyr aromatic), 7.10 (s, 1H, Trp aromatic), 7.15 (t, J = 7.2 Hz, 1H, Trp aromatic),
176
Experimental Section
7.31 (d, J = 8.1 Hz, 1H, Trp aromatic), 7.58 (d, J = 7.8 Hz, 1H, Trp aromatic), 8.31 (s, 1H,
Indole NH);
13
C NMR (100 MHz, CDCl3): δ = -4.5 (2C, CH3TBS), 17.1 (Ala CH3), 18.1 (quarternary C,
tBu (TBS)), 23.1 (Trp CH2), 25.6 (3C, tBu (TBS)), 28.3 (Boc tBu), 30.7 (NCH3), 40.3 (β-Tyr
CH2), 46.3 (Ala CH), 49.3 (β-Tyr CH), 51.7 (OCH3), 56.5 (Trp CH), 79.7 (quarternary C
Boc), 110.9, 111.0, 118.5, 119.3 (Trp aromatic), 120.1 (β-Tyr aromatic), 121.9, 122.0 (Trp
aromatic), 127.3 (β-Tyr aromatic), 133.3 (Tyr aromatic), 136.1 (aromatic), 154.9 (Boc CO),
155.5 (phenolic), 169.2 (Trp CO), 171.1 (β-Tyr CO), 174.3 (Ala CO);
HRMS (EI): calcd for C36H53N4O7Si [M+Na]+: 681.36780, found 681.36692.
Normethyl chondramide C (185)
OH
O
H
H
N
Me
N
N
O
O
O
N
H
O
To a solution of linear depsipeptide 193 (45 mg, 0.05 mmol) in CH2Cl2 (0.5 mL) was added
TFA (0.08 mL, 0.99 mmol) at 0 oC. The resulting mixture was stirred for 2 h at 0 oC; at this
point TLC showed the complete consumption of reactant. The solvent was removed in vacuo
and the residue dried by azeotropic removal of H2O with toluene. The crude material was used
for the next reaction without further purification. To a solution of crude amine salt in dry DMF
(50 mL) were added DIEA (0.04 mL, 0.20 mmol), HOBt (20 mg, 0.15 mmol) and TBTU (48
mg, 0.15 mmol) successively at room temperature. The solution was stirred at room
temperature for 18 h and then partitioned between ethyl acetate (50 mL) and water (50 mL).
The aqueous layer extracted with ethyl acetate (3 x 30 mL). The combined ethyl acetate layers
were washed successively with 5% aqueous KHSO4 solution, water, half saturated aqueous
NaHCO3 solution, brine, dried (MgSO4), filtered and concentrated in vacuo to furnish the
crude product. The crude product was purified by flash chromatogaphy (1:1 ethyl
Experimental Section
177
acetate/petroleum ether) to yield the pure cyclic depsipeptide (16 mg, 44%) as a colorless
solid.
To the above TBS protected cyclic depsipeptide (16 mg, 0.02 mmol) in THF (0.2 mL)
was added TBAF containing 5% water (1 M solution in THF, 0.04 mL, 0.04 mmol) at 0 oC.
The solution was stirred for 3 h at 0 oC. The mixture was concentrated in vacuo and the crude
material was purified by flash chromatography (7:3 ethyl acetate/petroleum ether) to provide
pure normethyl chondramide C (10 mg, 73%) as a colorless powder.
Rf = 0.35 (ethyl acetate);
[α]20D = +32.1 (c 0.137, CH2Cl2);
IR (film): υmax = 3360, 2950, 2850, 1710, 1510, 1170 cm-1;
1
H NMR (400 MHz, CDCl3): δ = 0.91 (d, J = 6.8 Hz, 3H, CH3CHCH2O), 1.01 (d, J = 6.8 Hz,
3H, Ala Me), 1.16 (d, J = 6.5 Hz, 3H, CH3CHCO), 1.63 (s, 3H, CH3C=C), 2.04-2.07 (m, 1H,
CH2CHCO), 2.17-2.24 (m, 1H, CH2CHCO), 2.44-2.50 (m, 1H, CHCO), 2.58-2.62 (m, 1H,
CHCH2O), 2.66-2.88 (m, 2H, β-Tyr CH2), 3.02 (s, 3H, NMe), 3.12 (dd, J = 15.0, 8.5 Hz, 1H,
Trp CH2), 3.30 (dd, J = 15.0, 7.5 Hz Trp CH2), 3.83 (dd J = 10.1, 5.6 Hz, 1H, CH2O), 3.91 (dd
J = 10.2, 4.7 Hz, 1H, CH2O), 4.65-4.70 (m, 1H, Ala CH), 4.93 (d, J = 8.0 Hz, olefinic), 5.205.26 (m, 1H, β-Tyr CH), 5.46 (t, J = 8.0 Hz, 1H, Trp CH), 6.43 (d, J = 5.8 Hz, 1H, Ala NH),
6.67 (d, J = 8.3 Hz, 2H, β-Tyr aromatic), 6.89 (d, J = 8.3 Hz, 2H, β-Tyr aromatic), 6.71 (s, 1H,
Trp aromatic), 6.92 (s, 1H, β-Tyr NH), 7.08 (t, J = 7.3 Hz, 1H, Trp aromatic), 7.15 (t, J = 7.5
Hz, 1H, Trp aromatic), 7.30 (d, J = 8.1 Hz, 1H, Trp aromatic), 7.54 (d, J = 7.8 Hz, 1H, Trp
aromatic), 8.26 (s, IndoleNH);
13
C NMR (100 MHz, CDCl3): δ = 17.6 (CH3CHCO), 18.0 (CH3C=C), 18.1 (CH3CHCH2O),
18.9 (Ala CH3), 23.9 (Trp CH2), 30.7 (NCH3), 31.8 (CHCH2O), 40.4 (CHCO), 41.1
(CH2CHCO), 42.5 (CH2β-Tyr), 45.7 (Ala CH), 49.7 (CH β-Tyr), 56.3 (Trp CH), 69.6 (CH2O),
110.2, 111.2 (Trp aromatic), 115.6 (β-TyrArmeta), 118.4, 119.5, 122.1, 122.5 (Trp aromatic),
125.7 (CH olefinic), 127.0 (β-Tyr Cipso), 127.2 (β-TyrArortho), 132.2 (Trp aromatic), 136.2
(olefinic quarternary), 155.6 (C-OH Ar), 169.5 (Trp CO), 170.9 (β-Tyr CO), 174.3 (Ala CO),
175.3 (CH2CHCO);
HRMS (EI): calcd for C34H42N4O6 [M+Na]+: 625.29966, found 625.29888.
178
Experimental Section
(2S–[(tert–Butoxycarbonyl)amino](4–{[tert-butyl(dimethyl)silyl]oxy}-αdiazoacetylbenzylamine (190)
OTBS
BocHN
N2
O
To a solution of acid 189 (1.00 g, 2.62 mmol) in ether (20 mL) were added triethylamine (0.41
mL, 2.91 mmol), and ethyl chlorocarbonate (0.28 mL, 2.62 mmol) successively at 0 oC. The
resulting reaction mixture was stirred at the same temperature for 1 h. Thereafter, a solution of
diazomethane in ether (20 mL, 0.127 M) was added and the mixture was stirred for 12 h at
room temperature. The reaction mixture was washed with water and the layers were separated.
The organic layer was then dried over anhydrous MgSO4, filtered, and concentrated in vacuo
to give a yellow colored solid. The crude product was almost pure on TLC, so the crude
diazoketone was used for next reaction without any purification.
Methyl (3R)-3-[(tert-Butoxycarbonyl)amino]-3-(4-{[tert-butyl(dimethyl)silyl]oxy}phenyl)
propanoate (186)
OTBS
O
H
N
Boc
OMe
To a solution of diazoketone 190 (400 mg, 0.98 mmol) in absolute methanol (10 mL) was
added dropwise a solution of of silver benzoate (23 mg, 0.1 mmol) in triethylamine (0.3 mL,
2.0 mmol) at -30 oC. The reaction mixture was slowly brought to room temperature in 3 h. The
reaction mixture was stirred additionally 1 h at room temperature. The mixture was filtered
Experimental Section
179
through a bed of celite and the filtrate concentrated in vacuo to get the crude product. The
crude product was purified by flash chromatography (1:4 ethyl acetate/petroleum ether) to
provide the pure ester 186 (340 mg, 80%) as a light yellow colored gel.
Rf = 0.40 (1:4 ethyl acetate/petroleum ether);
[α]20D = +36.1 (c 0.21, CH2Cl2);
IR (film): υmax = 3360, 2950, 2850, 1710, 1510, 1170 cm-1;
1
H NMR (400 MHz, CDCl3): δ = 0.11 (s, 6H, CH3TBS), 0.95 (s, 9H, tBu(TBS)), 1.41 (s, 9H,
Boc tBu), 2.76 (dd, J = 15.0, 5.9 Hz, 1H, CH2), 2.83-2.87 (m, 1H, CH2), 3.59 (s,3H, OCH3),
5.03 (br s, 1H, CH), 5.34 (br s, 1H, NH), 6.77 (d, J = 8.3 Hz, 2H, aromatic), 6.77 (d, J = 8.3
Hz, 2H, aromatic), 7.00 (d, J = 8.34 Hz, 2H, aromatic);
13
C NMR (100 MHz, CDCl3): δ = -4.5 (2C, CH3TBS), 18.2 (quarternary C, tBu (TBS)), 25.6
(3C, tBu (TBS)), 28.3 (Boc tBu), 40.9 (CH2), 51.7 (OCH3), 120.1, 127.2, 138.1 (aromatic),
155.0 (Boc CO), 155.1 (phenolic);
HRMS (EI): calcd for C21H35NO5Si [M+Na]+: 432.21767, found 432.21796.
N-(tert-Butoxycarbonyl)-L-alanyl-N-{(1R)-3-{[(2S,6S)-7-tert-butoxy-2,4,6-trimethyl-7oxohept-3-enyl]oxy}-3-oxo-1-[4-(1,1,2,2-tetramethylpropoxy)phenyl]propyl}-N-methylD-tryptophanamide (193)
OTBS
O
H
H
N
Me
N
O
N
O
O
H
N
Boc
O
OtBu
To a solution of tripeptide 184 (50 mg, 0.08 mmol) in 1,2-dichloroethane (1 mL) was added
trimethyltin hydroxide (68 mg, 0.38 mmol) at room temperature. The mixture was heated at 80
180
o
Experimental Section
C for 5 h. After cooling to room temperature, the reaction mixture was treated with 5%
aqueous KHSO4 until pH ~3 and extracted with ethyl acetate (3 x 4 mL). The combined ethyl
acetate layers were washed with brine (5 mL), dried (Na2SO4), filtered, and concentrated in
vacuo to give the crude acid which was used without further purification.
To a solution of crude acid, alcohol component 170 (20 mg, 0.08 mmol), and DMAP
(5 mg) in dry CH2Cl2 (1 mL) was added a solution of DCC (21 mg, 0.1 mmol) in CH2Cl2 (0.2
mL) at 0 oC. The resulting reaction mixture was stirred for 0.5 hour at 0 oC and then 12 h at
room temperature. The dicyclohexyl urea was filtered off and the precipitate was washed with
ether (3 x 4 mL). The filtrate was concentrated in vacuo to provide the crude product. The
crude product was purified by flash chromatography (1:1 ethyl acetate/petroleum ether) to
furnish the pure product 193 (50 mg, 72% over two steps) as a colorless gel.
Rf = 0.44 (1:1 ethyl acetate/petroleum ether);
[α]20D = +10.2 (c 0.50, CH2Cl2);
IR (film): υmax = 3336, 2930, 2850, 2300, 1730, 1685, 1515, 1260, 1160 cm-1;
1
H NMR (400 MHz, CDCl3): δ = 0.15 (s, 6H, CH3TBS), 0.86 (d, J = 7.6 Hz, CH3CHCH2O),
0.88 (d, J = 7.6 Hz, 3H, Ala CH3), 0.95 (s, 9H, tBu(TBS)), 1.02 (d, J = 6.8 Hz,
CH3CHCO2tBu), 1.39, 1.41 (2s, 18H, Boc tBu, tBu), 1.57 (s, 3H, olefinic CH3), 1.94 (dd, J =
13.6, 7.6 Hz, 1H, CH2CHCO2tBu), 2.28 (dd, J = 13.6, 7.6 Hz, 1H, CH2CHCO2tBu), 2.41-2.48
(m, 1H, CHCO2tBu), 2.58-2.64 (m, 1H, CHCH2O), 2.74 (dd, J = 15.5, 5.9 Hz, 1H, β-Tyr
CH2), 2.86 (dd, J = 16.6, 8.2 Hz, 1H, β-Tyr CH2), 2.90 (s, 3H, NCH3), 3.20 (dd, J = 15.7, 10.6
Hz, 1H, Trp CH2), 3.43 (dd, J = 15.8, 5.2 Hz, 1H, Trp CH2), 3.69-3.74 (m, 1H, CH2O), 3.83
(dd, J = 10.1, 6.6 Hz, 1H, CH2O), 4.36 (qn, J = 6.7 Hz, 1H, Ala CH), 4.88 (d, J = 9.4 Hz, 1H,
CH olefinic), 5.35 (q, J = 6.9 Hz, 1H, β-Tyr CH), 5.59 (dd, J = 9.9, 5.6 Hz, Trp CH), 6.71 (d, J
= 8.3 Hz, 2H, β-Tyr aromatic), 6.93 (s, 1H, Trp aromatic), 7.01 (d, J = 8.1 Hz, 1H, β-Tyr NH),
7.07-7.09 (m, 2H, β-Tyr aromatic), 7.14 (t, J = 7.3 Hz, 1H, Trp aromatic), 7.30 (d, J = 8.1 Hz,
1H, Trp aromatic), 7.36 (t, J = 8.1 Hz, 1H, Trp aromatic), 7.57 (d, J = 7.8 Hz, 1H, Trp
aromatic), 8.29 (s, 1H, Indole NH);
13
C NMR (100 MHz, CDCl3): δ = -4.5 ( 2C, CH3TBS), 16.7 (CH3CHCO2tBu), 17.0
(quarternary C, tBu (TBS)), 17.4 (CH3CHCH2O), 18.1 (Ala CH3), 23.2 (Trp CH2), 25.6 (3C,
tBu (TBS)), 28.0, 28.3 (6C, tBu, Boc tBu), 30.8 (NCH3), 31.9 (CHCH2O), 38.6 (CHCO2tBu),
40.5 (β-Tyr CH2), 43.8 (CH2CHCO2tBu), 46.6 (Ala CH), 49.4 (β-Tyr CH), 56.6 (Trp CH),
Experimental Section
181
68.8 (CH2O), 79.7, 79.8 (quarternary C Boc, tBu), 111.0, 118.5, 119.3 (Trp aromatic), 120.0
(β-Tyr aromatic), 121.9, 122.0 (Trp aromatic), 127.4 (β-Tyr aromatic), 128.2 (aromatic), 129.5
(aromatic), 133.3 (Tyr aromatic), 134.2, 134.8, 136.1 (aromatic), 154.9 (Boc CO), 155.6
(phenolic), 169.2 (Trp CO), 170.7 (β-Tyr CO), 174.3 (Ala CO), 175.7 (CO2tBu);
HRMS (EI): calcd for C49H74N4O9Si [M+H]+: 891.52978, found 891.53001.
182
Experimental Section
(2S)-3-(3-{[(tert-Butoxycarbonyl)amino]methyl}phenyl)-2-methylpropanoic acid (194)
O
HO
NHBoc
To a solution of oxazolidinone 204 (300 mg, 0.66 mmol) in THF (10 mL) were added 30 wt%
H2O2 (0.28 mL, 2.64 mmol) and 0.4 N lithium hydroxide monohydrate solution (3.3 mL, 1.32
mmol) successively at 0 oC. The resulting reaction mixture was stirred for 3 h at 0 oC before it
was treated with saturated aqueous Na2SO3 solution (6 mL) and saturated aqueous NaHCO3
solution (6 mL) at 0 oC. The mixture was extracted with dichloromethane (2 x 10 mL) to
remove the chiral auxiliary. The aqueous layer was acidified to pH~3 by the addition of 1 N
HCl and extracted with ethyl acetate (3 x 15 mL). The combined ethyl acetate layers were
dried over Na2SO4, filtered and concentrated in vacuo to furnish the almost pure amino acid
194. But due to the presence of amino acid in the dichloromethane layer, both the products
from the ethyl acetate and dichloromethane layers were combined and purified using flash
chromatography (2:3 ethyl acetate/petroleum ether) to afford the pure acid 194 (155 mg, 80%)
as a colorless gel.
Rf = 0.44 (2:3 ethyl acetate/petroleum ether);
[α]20D = +18.3 (c 0.96, CH2Cl2);
IR (film): υmax = 3410, 2965, 2935, 1780, 1700, 1515, 1380 cm-1;
1
H NMR (400 MHz, CDCl3): δ = 1.19 (d, J = 7.1 Hz, 3H, CH3), 1.48 (s, 9H, tBu), 2.68 (dd,
13.3, 8.0 Hz, 1H, CH2CH), 2.77 (td, J = 14.0, 6.8 Hz, 1H, CHCH3), 3.08 (dd, J = 13.4, 6.3 Hz,
1H, CH2CH), 4.31 (d, J = 5.1 Hz, 2H, CH2N), 4.89 (br s, 1H, NH), 7.10-7.16 (m, 3H,
aromatic), 7.28 (d, J = 6.8 Hz, 1H, aromatic);
13
C NMR (100 MHz, CDCl3): δ = 16.5 (CH3), 28.4 (3C, tBu), 39.2 (CH2CH), 41.1 (CHCH3),
44.6 (CH2NH), 79.8 (Boc quarternary), 125.6, 128.0, 128.7, 139.0, 139.5 (aromatic), 155.3
(Boc C=O), 181.3 (CO2H);
HRMS (EI): calcd for C16H23NO4 [M+Na]+: 316.15193, found 316.15113.
Experimental Section
183
(2S)-3-[3-({[tert-Butyl(dimethyl)silyl]oxy}methyl)phenyl]-2-methylpropanoic acid (195)
O
HO
OTBS
To a solution of crude unprotected hydroxy acid 205 (50 mg, 0.26 mmol) in DMF (3 mL)
imidazole (50 mg, 0.78 mmol) and TBDMS-Cl (90 mg, 0.57 mmol) were added successively
at room temperature. The reaction mixture was stirred for 12 h at room temperature. H2O (5
mL) was added to reaction mixture and stirring was continued for 30 min. The resulting
mixture was extracted with diethyl ether (3 x 5 mL). The combined ether layers were
successively washed with 1 N HCl (5 mL), saturated aq. NaHCO3 solution (5 mL) and then
with brine (5 mL). The dried (Na2SO4) layer was filtered and concentrated in vacuo to give the
crude product. This adduct was dissolved in THF (1 mL) and 1 M K2CO3 (1 mL) was added to
the solution at room temperarure and the mixture stirred for 45 min at room temperature. The
resulting mixture was acidified to pH~3 by adding 1 N HCl. The mixture was extracted with
ethyl acetate (3 x 3 mL). The combined ethyl acetate layers were dried on Na2SO4, filtered and
evaporated to get the crude TBS protected hydroxy acid which was purified by flash
chromatography (1:3 ethyl acetate/petroleum ether) providing the pure hydroxy acid 195 (55
mg, 70%) as a colorless gel.
Rf = 0.50 (1:3 ethyl acetate/petroleum ether);
[α]20D = +22.9 (c 1.18, CH2Cl2);
IR (film): υmax = 3740, 3180, 2930, 2850, 1700, 1520, 1170, 840 cm-1;
1
H NMR (400 MHz, CDCl3): δ = 0.10 (s, 6H, CH3TBS), 0.95 (s, 9H, tBu(TBS)), 1.17 (d, J =
7.1 Hz, 3H, CHCH3), 2.66 (dd, J = 13.3, 8.2 Hz, 1H, CH2CH), 2.71-2.81 (m, 1H, CHCO2H),
3.10 (dd, J = 13.4, 6.3 Hz, 1H, CH2CH), 4.73 (s, 2H, CH2OTBS), 7.07 (d, J = 7.3 Hz, 1H,
aromatic), 7.15 (s, 1H, aromatic), 7.19 (d, J = 7.8 Hz, 1H, aromatic), 7.26 (t, J = 7.5 Hz, 1H,
aromatic);
13
C NMR (100 MHz, CDCl3): δ = -5.3 (2C, CH3TBS), 16.4 (CHCH3), 18.4 (quarternary C,
tBu (TBS)), 25.9 (3C, tBu (TBS)), 39.2 (CH2CHCO2), 64.9 (CH2OTBS), 124.20, 126.7, 127.6,
128.3, 138.9, 141.5 (aromatic), 182.5 (CO2H);
184
Experimental Section
HRMS (EI): calcd for C17H28O3Si [M-H]-: 307.17349, found 307.17354.
3-{(2S)-3-[(4R)-4-Benzyl-2-oxo-1,3-oxazolidin-3-yl]-2-methyl-3-oxopropyl}benzonitrile
(196)
O
O
O
CN
N
Bn
To a solution of crude alkylation product 198 (400 mg, 0.99 mmol) in DMF (4 mL) was added
copper(I) cyanide (100 mg, 1.10 mmol) at room temperature. The reaction mixture was heated
at 100 oC for 12 h. The hot reaction mixture was poured in a solution of FeCl3 (100 mg) in 4
mL of water. The resulting mixture was extracted with ethyl acetate (3 x 10 mL). The
combined organic layers were washed with brine (10 mL), dried (Na2SO4), filtered and
evaporated.
The
crude
product
was
purified
by
flash
chromatography
(1:4
ethylacetate/petroleum ether) to furnish the pure nitrile 196 (200 mg, 57%) as a colorless gel.
Rf = 0.38 (1:4 ethyl acetate/petroleum ether);
1
H NMR (400 MHz, CDCl3): δ = 1.12 (d, J = 6.8 Hz, 3H, CH3), 2.55-2.58 (m, 1H, benzylic
H), 2.60-2.63 (m, 1H, benzylic H), 3.04 (dd, J = 13.4, 3.3 Hz, 1H, CH2Ph), 3.14 (dd, J = 13.4,
6.8 Hz, 1H, CH2Ph), 3.95-4.04 (m, 1H, CHCH3), 4.09 (dd, J = 9.1, 2.8 Hz, 1H, CH2O), 4.15 (t,
J = 8.5 Hz, 1H, CH2O), 4.59-4.65 (m, 1H, NCH), 7.00-7.01 (m, 2H, aromatic), 7.17-7.25 (m,
3H, aromatic), 7.34 (t, J = 7.7 Hz, 1H, aromatic), 7.45 (d, J = 7.6 Hz, 1H, aromatic), 7.50 (d, J
= 7.6 Hz, 1H, aromatic), 7.53 (s, 1H, aromatic);
13
C NMR (100 MHz, CDCl3): δ = 16.5 (CH3), 37.7 (PhCH2), 39.1 (CHCH3), 39.5 (benzylic),
55.1 (NCH), 66.0 (OCH2), 112.4 (aromatic), 118.8 (CN), 127.4, 128.9, 129.2, 129.3, 130.3,
132.8, 140.7 (aromatic), 153.0 (NCO2), 175.6 (CO).
(4R)-4-Benzyl-3-{(2S)-3-[3-(bromomethyl)phenyl]-2-methylpropanoyl}-1,3-oxazolidin-2one (198)
Experimental Section
185
O
O
O
N
Br
Bn
To a solution of propionyl oxazolidinone 100 (500 mg, 2.15 mmol) in dry THF (10 mL) was
added a 2.0 M solution of NaHDMS in THF (1.60 mL, 3.21 mmol) at -78 oC. The reaction
mixture was stirred for 2 h at -78 oC. A solution of 3-brombenzyl bromide (1.34 g, 5.36 mmol)
in THF (4 mL) was added dropwise at -78 oC. The reaction mixture was stirred for 6 h at -78
o
C and brought to room temperature by removing the cooling bath. The reaction mixture was
treated with saturated aqueous NH4Cl solution (10 mL) and partially concentrated in vacuo.
The aqueous layer was extracted with ethyl acetate (3 x 10 mL). The combined ethyl acetate
layers were washed with brine (10 mL), dried (Na2SO4), filtered and concentrated in vacuo.
The crude product was purified by flash chromatography (1:4 ethyl acetate/petroleum ether) to
afford the desired alkylated product 198, which conatained about 15% of the chiral auxiliary
100 (determined by NMR).
Rf = 0.56 (1:3 ethyl acetate/petroleum ether);
13
C NMR (100 MHz, CDCl3): δ = 16.8 (CH3), 37.6 (PhCH2), 39.3 (CHCH3), 39.5 (benzylic),
55.0 (NCH), 65.9 (OCH2), 122.3, 127.3, 128.0, 128.8, 129.3, 129.9, 132.2, 135.0, 141.5
(aromatic), 152.9 (NCO2), 176.0 (CO).
Methyl (2S)-3-(3-cyanophenyl)-2-methylpropanoate 199
O
MeO
CN
Methylmagnesium bromide (3 M solution in THF, 0.20 mL, 0.55 mmol) was added to precooled (0 oC) absolute methanol (4.5 mL). After stirring for 20 minutes, this solution was
added via cannula to a solution of oxazolidinone 196 in absolute methanol (4.5 mL) at 0 oC.
The resulting reaction mixture was stirred for 6 h at 0 oC. The reaction mixture was acidified
186
Experimental Section
with 1 N HCl until pH~3 and concentrated in vacuo to remove the methanol. The resulting
aqueous layer was extracted with diethyl ether (3 x 5 mL). The combined organic layers were
washed with brine, dried (Na2SO4), filtered and concentrated in vacuo to obtain the crude
product which was purified using flash chromatography (1:3 ethyl acetate/petroleum ether) to
give the pure methylester 199 (30 mg, 52%) as a colorless gel.
Rf = 0.53 (1:3 ethyl acetate/petroleum ether);
1
H NMR (400 MHz, CDCl3): δ = 1.16 (d, J = 6.8 Hz, 3H, CH3), 2.68-2.75 (m, 2H, benzylic
H), 2.99-3.06 (m, 1H, CHCH3), 3.62 (s, 3H, OCH3), 7.35-7.41 (m, 2H, aromatic), 7.45 (s, 1H,
aromatic), 7.48-7.51 (m, 1H, aromatic);
13
C NMR (100 MHz, CDCl3): δ = 16.9 (CH3), 39.1 (benzylic), 41.1 (CHCH3), 55.7 (OCH3),
112.4 (aromaric), 118.8 (CN), 129.2, 130.2, 132.4, 133.5, 140.8 (aromatic), 175.6 (CO2Me).
Methyl(2S)-3-(3-{[(tert-butoxycarbonyl)amino]methyl}phenyl)-2-methylpropanoate (200)
O
MeO
NHBoc
A solution of cyano compound 199 (25 mg, 0.11 mmol) in methanol (0.5 mL) was added to a
suspension of 10% Pd/C (10 mg) and formic acid (0.06 mL) in ethanol (0.5 mL). The resulting
mixture was shaken under hydrogen atmosphere at around 2 bar pressure for 14 h. The
reaction mixture was filtered through a celite bed and the bed washed with ethyl acetate (3 x 1
mL). The combined organic layers were evaporated to give the crude amine formate salt (28
mg). The compound was used for the protection without further purification.
To the crude amine salt in dioxane/H2O (2:1) (0.6 mL) was added 1 N NaOH (0.16
mL, 0.16 mmol) followed by Boc anhydride (27 mg, 0.121 mmol) at room temperature. The
reaction mixture was stirred for 5 h at room temperature. The reaction mixture was acidified
with 5% aq. KHSO4 to pH~3. The resulting mixture was extracted with ethyl acetate (3 x 4
mL). The combined ethyl acetate layers were washed with brine (4 mL), dried (Na2SO4),
Experimental Section
187
filtered and concentrated in vacuo to give the crude product which was purified using flash
chromatography (15:85 ethyl acetate/petroleum ether) to afford the pure amino acid
methylester 200 (20 mg, 59% over two steps) as a colorless gel.
Rf = 0.44 (15:85 ethyl acetate/petroleum ether);
1
H NMR (400 MHz, CDCl3): δ = 1.13 (d, J = 6.8 Hz, 3H, CH3), 1.45 (s, 9H, tBu), 2.63 (dd, J
= 13.1, 7.8 Hz, 1H, benzylic H), 2.71 (td, J = 14.0, 6.7 Hz, 1H, CHCH3), 3.00 (dd, J = 13.1,
6.6 Hz, 1H, benzylic H), 3.63 (s, 3H, OCH3), 4.27 (d, J = 5.6 Hz, 2H, CH2NH), 4.81 (br s, 1H,
NH), 7.04 (d, J = 7.8 Hz, 1H, aromatic), 7.05 (s, 1H, aromatic), 7.23 (t, J = 7.7 Hz, 1H,
aromatic);
13
C NMR (100 MHz, CDCl3): δ = 16.7 (CH3), 28.4 (3C, tBu), 39.6 (benzylic), 41.3 (CHCH3),
44.6 (CH2NH), 51.6 (OCH3), 125.5, 127.9, 128.0, 128.6, 139.0, 139.8 (aromatic), 155.8 (Boc
CO), 176.5 (CO2Me).
(4R)-3-{(2S)-3-[3-(azidomethyl)phenyl]-2-methylpropanoyl}-4-benzyl-1,3-oxazolidin-2one (202)
O
O
O
N
N3
Bn
To a solution of the mono alkylation product 202 (2.00 g, 4.80 mmol) in ethanol (30 mL) was
added sodium azide (650 mg, 10.10 mmol) at room temperature. The reaction mixture was
heated to reflux for 2 h. The reaction mixture was cooled to room temperature, treated slowly
with water and concentrated in vacuo. The resulting mixture was dissolved in ethyl acetate (30
mL) and washed with water (2 x 10 mL). The organic layer was dried over Na2SO4, filtered
and concentrated in vacuo to obtain almost pure azide (1.55 g, 85%) as a pale yellow gel.
Rf = 0.44 (1:3 ethyl acetate/petroleum ether);
[α]20D = -19.0 (c 1.07, CH2Cl2);
IR (film): υmax = 2975, 2930, 2850, 2098, 1780, 1697, 1100 cm-1;
188
1
Experimental Section
H NMR (400 MHz, CDCl3): δ = 1.11 (d, J = 6.6 Hz, 3H, CH3), 2.48 (dd, J = 13.4, 9.4 Hz,
1H, benzylic H), 2.60 (dd, J = 13.1, 7.6 Hz, 1H, CHCH2Ph), 3.01 (dd, J = 13.4, 9.4 Hz, 1H,
benzylic H), 3.09 (dd, J = 13.3, 7.2 Hz, 1H, CHCH2Ph), 4.01-4.10 (m, 3H, CHCH3, CH2O),
4.22 (s, 2H, CH2N3), 4.55-4.61 (m, 1H, NCH), 6.99 (d, J = 7.6 Hz, 1H, aromatic), 7.00 (s, 1H,
aromatic), 7.09 (d, J = 7.1 Hz, 1H, aromatic), 7.17-7.23 (m, 6H, aromatic);
13
C NMR (100 MHz, CDCl3): δ = 16.6 (CH3), 37.6 (PhCH2), 39.5 (CHCH3), 39.6 (benzylic),
54.6 (CH2N3), 55.1 (NCH), 65.9 (OCH2), 126.3, 127.2, 128.8, 129.2, 129.3, 135.1, 135.3,
139.9 (aromatic), 153.0 (NCO2), 176.2 (CO);
HRMS (EI): calcd for C21H22N4O3Si [M+Na]+: 401.15841, found 401.15804.
(4R)-4-Benzyl-3-{(2S)-3-[3-(bromomethyl)phenyl]-2-methylpropanoyl}-1,3-oxazolidin-2one (203)
O
O
O
N
Br
Bn
To a solution of diisopropylamine (4.80 mL, 33.5 mmol) in absolute THF (60 mL) was added
n-BuLi (2.5 M solution in hexane, 13.40 mL, 33.5 mmol) at 0 oC. The resulting mixture was
stirred for 20 min at 0 oC and cooled to -78 oC. A solution of propionyl oxazolidinone 100
(6.00 g, 25.75 mmol) in absolute THF (12 mL) was added dropwise to the above solution of
LDA. The reaction mixture was stirred for 2 h at -78 oC. A solution of α,α’-dibromo m-xylene
104 (17.0 g, 64.4 mmol) in absolute THF (20 mL) was added dropwise to the reaction mixture
at -78 oC. The resulting reaction mixture was stirred for 4 h at -78 oC and slowly warmed to
room temperature in 4 h. The reaction mixture was treated with saturated aqueous NH4Cl
solution (30 mL) and then the mixture was partially concentrated in vacuo. The resulting
aqueous layer was extracted with ethyl acetate (3 x 30 mL). The combined ethyl acetate layers
were washed with saturated aqueous NaHCO3 (30 mL), brine (30 mL), dried (Na2SO4),
filtered, and concentrated in vacuo to give the crude product. Most of the excess α,α’-dibromo
m-xylene was removed by dissolving the crude product in hexane (50 mL). The remaining
Experimental Section
189
crude product was purified using flash chromatography (1:3 ethyl acetate/petroleum ether) to
give the pure mono alkylation product 203 (6.20 g, 58%) as a colorless gel.
Rf = 0.50 (1:3 ethyl acetate/petroleum ether);
[α]20D = -16.8 (c 1.23, CH2Cl2);
IR (film): υmax = 2975, 2905, 2850, 1780, 1697, 1385, 1200 cm-1;
1
H NMR (400 MHz, CDCl3): δ = 1.12 (d, J = 6.8 Hz, 3H, CH3), 2.48 (dd, J = 13.7, 9.6 Hz,
1H, benzylic H), 2.60 (dd, J = 13.3, 7.5 Hz, 1H, CH2Ph), 2.99 (dd, J = 13.4, 3.3 Hz, 1H,
benzylic H), 3.06 (dd, J = 13.3, 7.5 Hz, 1H, CH2Ph), 4.02 (dd, J = 9.1, 3.0 Hz, 1H, CH2O),
4.05-4.11 (m, 2H, CHCH3, CH2O), 4.39 (s, 2H, CH2Br), 4.55-4.61 (m, 1H, NCH), 6.99 (d, J =
7.6 Hz, 1H, aromatic), 7.00 (s, 1H, aromatic), 7.14-7.20 (m, 6H, aromatic), 7.24 (s, 1H,
aromatic);
13
C NMR (100 MHz, CDCl3): δ = 16.8 (CH3), 33.5 (CH2Br), 37.7 (PhCH2), 39.4 (CHCH3),
39.6 (benzylic), 55.1 (NCH), 65.9 (OCH2), 127.2, 127.3, 128.6, 129.3, 129.5, 129.8, 129.9,
135.1, 137.8, 139.8 (aromatic), 153.0 (NCO2), 176.3 (CO);
HRMS (EI): calcd for C21H22BrNOSi [M+Na]+: 438.06753, found 438.06839.
tert-Butyl 3-{(2S)-3-[(4R)-4-benzyl-2-oxo-1,3-oxazolidin-3-yl]-2–methyl–3-oxopropyl}
benzylcarbamate (204)
O
O
O
N
NHBoc
Bn
A solution of azide 202 (400 mg, 1.06 mmol) in ethanol (5 mL) was added to 10% Pd/C (40
mg) in a hydrogenation vessel. The reaction mixture was shaken under hydrogen atmosphere
at around 2 bar pressure for 20 h. The reaction mixture was filtered through a bed of celite and
the celite bed was washed with ethyl acetate (5 mL). The filtrate was concentrated in vacuo to
get the crude amine which was used for the protection reaction without further purification. To
a solution of crude amine in dioxane/H2O (2:1) (6 mL) was added 1 N NaOH (2.2 mL, 2.2
mmol) followed by the addition of Boc-anhydride (272 mg, 1.25 mmol) at room temperature.
190
Experimental Section
The reaction mixture was stirred at room temperature for 2 h. The reaction mixture was
acidified to pH~3 using 5% aq. KHSO4 and the mixture was extracted with ethyl acetate (3 x
10 mL). The combined ethyl acetate layers were washed with brine (15 mL), dried (Na2SO4),
filtered and concentrated in vacuo to give the crude product which was purified by flash
chromatography (1:3 ethyl acetate/petroleum ether) providing the pure product 204 (330 mg,
65%) as a colorless gel.
Rf = 0.53 (1:3 ethyl acetate/petroleum ether);
[α]20D = -15.9 (c 0.97, CH2Cl2);
IR (film): υmax = 3410, 2965, 2935, 2850, 1780, 1700, 1515, 1380, 1240 cm-1;
1
H NMR (400 MHz, CDCl3): δ = 1.14 (d, J = 6.8 Hz, 3H, CH3), 1.42 (s, 9H, tBu), 2.55 (dd, J
= 13.5, 9.5 Hz, 1H, benzylic H), 2.62 (dd, J = 13.3, 8.0 Hz, 1H, CH2Ph), 3.04-3.08 (m, 1H,
benzylic H), 3.08-3.14 (m, 1H, CH2Ph), 4.03-4.10 (m, 2H, CHCH3, CH2O), 4.15 (t, J = 8.5 Hz,
1H, CH2O), 4.24 (d, J = 5.3 Hz, 2H, CH2NH), 4.61-4.67 (m, 1H, NCH), 4.76 (br s, 1H, NH),
7.03 (d, J = 6.3 Hz, 1H, aromatic), 7.04 (s, 1H, aromatic), 7.11 (d, J = 7.3 Hz, 1H, aromatic),
7.15-7.17 (m, 2H, aromatic), 7.21-7.29 (m, 4H, aromatic);
13
C NMR (100 MHz, CDCl3): δ = 16.6 (CH3), 28.4 (3C, tBu), 37.7 (PhCH2), 39.5 (benzylic),
39.7 (CHCH3), 44.6 (CH2NH), 55.1 (NCH), 65.9 (OCH2), 79.4 (Boc quarternary), 125.6,
127.3, 128.3, 128.5, 128.6, 128.9, 129.4, 135.1, 138.9, 139.6 (aromatic), 153.0 (NCO2), 155.8
(Boc CO), 176.5 (CO);
HRMS (EI): calcd for C26H32N2O5Si [M+Na]+: 475.22034, found 475.22056.
(2S)-3-[3-(Hydroxymethyl)phenyl]-2-methylpropanoic acid (205)
O
HO
OH
To a solution of monoalkylation product 203 (200mg, 0.48 mmol) in THF (10 mL) were added
30 wt% H2O2 (0.33 mL, 2.88 mmol) and 0.4 N lithium hydroxide monohydrate solution (3.60
mL, 1.44 mmol) successively at 0 oC. The resulting reaction mixture was stirred for 7 h at 0
Experimental Section
o
191
C. The reaction mixture was then treated with saturated aqueous Na2SO3 solution (8 mL) and
saturated aqueous NaHCO3 solution (8 mL) at 0 oC. The mixture was extracted with
dichloromethane (2 x 10 mL) to remove the chiral auxiliary. The aqueous layer acidified to
pH~2 by the addition of 6 N HCl. The aqueous layer was carefully extracted with ethyl acetate
(4 x 15 mL). The combined ethyl acetate layers were dried over Na2SO4, filtered and
concentrated in vacuo to afford the crude hydroxy acid 205 (65 mg, 70%) as a colorless oil.
The crude mixture was used further without any purification.
tert-Butyl({(3R,4R,5R)-4-[(4-methoxybenzyl)oxy]-3,5-dimethylhept-6enyl}oxy)dimethylsilane (213)
OPMB
OTBS
To a solution of methyltriphenylphosphonium iodide (1.23 g, 3.05 mmol) in THF (60 mL) was
added a 2.5 M solution of n-BuLi (1.50 mL, 3.56 mmol) in hexane at 0 oC. The resulting
mixture was stirred for 30 min at 0 oC and a solution of aldehyde 232 (800 mg, 2.03 mmol) in
THF (6 mL) was added at 0 oC. The mixture was stirred for further 2 h at 0 oC. The reaction
mixture was treated with pH 7 phosphate buffer (30 mL). The resulting aqueous layer was
extracted with CH2Cl2 (3 x 30 mL). The combined organic layers were washed with brine,
dried (Na2SO4), filtered and concentrated. The crude product was purified by flash
chromatography (5:95 ethyl acetate/petroleumether) to obtain the pure alkene 213 (590 mg,
74%) as a colorless gel.
Rf = 0.55 (5:95 ethyl acetate/petroleum ether);
[α]25D = +5.24 (c 1.01, CH2Cl2);
IR (film): νmax = 3070, 2950, 2860, 1620, 1515, 1460, 1250, 1170 cm-1;
1
H NMR (400 MHz, CDCl3): δ = 0.03 (s, 6H, Si(CH3)3), 0.88 (s, 9H; tBu), 0.96 (d, J = 6.8
Hz, 3H, CH3), 1.07 (d, J = 6.8 Hz, 3H, CH3CHCH=CH2), 1.32-1.41 (m, 1H,
CHCH2CH2OTBS), 1.79-1.92 (m, 2H, CH2CH2OTBS), 2.43-2.51 (m, 1H, CHCH=CH2), 3.06
192
Experimental Section
(dd, J = 6.6, 4.6 Hz, 1H, CHOPMB), 3.56-3.62 (m, 1H, CH2OTBS), 3.65-3.71 (m, 1H,
CH2OTBS), 3.79 (s, 3H, OCH3), 4.49 (d, J = 10.9 Hz, 1H, CH2Ph), 4.52 (d, J = 10.9 Hz, 1H,
CH2Ph), 4.96 (d, J = 10.4 Hz, 1H, olefinic CH2 cis), 5.04 (d, J = 17.2 Hz, 1H, olefinic CH2
trans), 5.81 (ddd, J = 17.3, 10.2, 8.1 Hz, 1H, CH olefinic), 6.86 (d, J = 8.6 Hz, 2H, aromatic),
7.27 (d, J = 8.8 Hz, 2H, aromatic);
13
C NMR (100 MHz, CDCl3): δ = -5.31, -5.28 (2C, Si(CH3)3), 15.9 (CH3CHCH2), 17.2
(CH3CHCH=CH2), 18.3 (TBS quarternary), 26.0 (3C, tBu), 32.5 (CHCH2OH), 34.5
(CH2CH2OTBS), 40.9 (CHCH2CH2OTBS), 55.3 (OCH3), 61.6 (CH2CH2OTBS), 74.5
(CH2Ph), 88.0 (CHOPMB), 113.6 (aromatic), 113.8 (olefin CH2), 129.1 (aromatic) 131.3 (Cipso
aromatic), 142.5 (olefin CH), 159.0 ppm (phenolic);
HRMS (EI): calcd for C23H40O3Si [M+Na]+: 415.26389, found 415.26386.
(4S)-4-Isopropyl-3-[(2R)-2-methylpent-4-enoyl]-5,5-diphenyl-1,3-oxazolidin-2-one (217)
O
O
O
N
Ph Ph
To a solution of auxiliary 216 (14.0 g, 41.54 mmol) in dry THF (90 mL) was added a 2.0 M
solution of NaHDMS (25 mL, 50.0 mmol) at -78 oC. The reaction mixture was stirred for 2 h
at -78 oC. Allylbromide (20.0 mL, 207.7 mmol) was added dropwise to the above mixture over
20 min at -78 oC. The resulting reaction mixture was stirred for 2 h at -78 oC and slowly
brought to room temperature by switching off the cooling machine and stirred overnight. The
reaction mixture was treated with saturated aqueous NH4Cl solution (50 mL) and diluted with
diethyl ether (100 mL). The layers were separated and the aqueous layer extracted with ether
(3 x 50 mL). The combined ether layers were washed with saturated aqueous NaHCO3
solution (50 mL), brine (50 mL), dried (Na2SO4), filtered and evaporated to obtain the almost
pure alkylated product as a pale yellow solid which was used for the next step without
purification.
Experimental Section
193
(2R)-2-Methylpent-4-en-1-ol (218)
OH
To a solution of the alkylation product 217 (41.54 mmol) in THF (800 mL) was added a
solution of NaBH4 (9.40 g, 249.3 mmol) in H2O (200 mL) at 0 oC. The resulting reaction
mixture was stirred for 12 h at room temperature. The reaction mixture was treated with
saturated aqueous NH4Cl solution (100 mL) at 0 oC and stirred for 1 h at 0 oC. The layers were
separated and the aqueous layer was extracted with ether (2 x 200 mL). The combined organic
layers were washed with saturated aqueous NaHCO3 solution (200 mL), brine (200 mL), dried
(Na2SO4), filtered and concentrated in vacuo to give the crude alcohol. As the chiral auxiliary
is insoluble in ether, the chiral auxiliary was removed by filtration and the remaining crude
product purified by flash chromatography (1:1 ether/petroleum ether) to deliver the pure
alcohol 218 (3.5 g, 90% two steps) as a light yellow colored oil.
Rf = 0.45 (1:1 ether/petroleum ether);
1
H NMR (400 MHz, CDCl3): δ = 0.90 (d, J = 6.6 Hz, 3H, CH3), 1.65-1.77 (m, 1H, CHCH3),
1.91 (ddd, J = 14.1, 7.3, 7.1 Hz, 1H, CHCH2), 2.12-2.19 (m, 1H, CHCH2), 3.42 (dd, J = 10.5,
5.4 Hz, 1H, CH2O), 3.49 (dd, J = 10.8, 5.4 Hz, 1H, CH2O), 4.99 (d, J = 9.1 Hz, 1H, cis H),
5.02 (d, J = 15.4 Hz, 1H, trans H), 5.73 (CH olefinic);
13
C NMR (100 MHz, CDCl3): δ = 16.3 (CH3), 35.5 (CHCH2), 37.8 (CHCH3), 67.8 (OCH2),
116.0 (olefinic CH2), 136.9 (olefinic CH).
(2S,3S,4R)-6-{[tert-Butyl(dimethyl)silyl]oxy}-3-[(4-methoxybenzyl)oxy]-2,4dimethylhexan-1-ol (223)
HO
OPMB
OTBS
194
Experimental Section
To a solution of acetal 224 (1.20 g, 3.04 mmol) in CH2Cl2 (30 mL) was added a 1 M solution
of DIBAL-H in hexane (9.10 mL, 9.10 mmol) at 0 oC. The reaction mixture was stirred for 4 h
at 0 oC and then treated with saturated sodium potassium tartarate solution (20 mL) at 0 oC.
The solution was stirred vigorously for 1 h at room temperature and the layers were separated.
The aqueous layer was extracted with CH2Cl2 (3 x 20 mL). The combined organic layers were
washed with water (20 mL) and then with brine. The organic layer was dried on Na2SO4,
filtered and concentrated in vacuo to give the crude product which on purification by flash
chromatography (1:3 ethyl acetate/petroleum ether) led the pure alcohol 223 (940 mg, 78%) as
a colorless oil.
Rf = 0.40 (1:3 ethyl acetate/petroleum ether);
[α]20D = -4.7 (c 1.08, CH2Cl2);
IR (film): νmax = 3430, 2935, 2850, 1620, 1515, 1460, 1250, 1090 cm-1;
1
H NMR (400 MHz, CDCl3): δ = -0.03 (s, 6H, Si(CH3)3), 0.82 (s, 9H; tBu), 0.86 (d, J = 6.8
Hz, 6H, 2CH3), 1.22-1.33 (m, 1H, CHCH2CH2), 1.78-1.89 (m, 2H, CH2CH2OTBS), 2.13 (br s,
1H, CHCH2OH), 3.25 (dd, J = 6.4, 3.7 Hz, 1H, CHOPMB), 3.45-3.51 (m, 2H, CH2OTBS),
3.55-3.59 (m, 1H, CH2OH), 3.62-3.66 (m, 1H, CH2OH), 3.70 (s, 3H, OCH3), 4.40 (d, J = 10.9
Hz, 1H, CH2Ph), 4.48 (d, J = 10.9 Hz, 1H, CH2Ph), 6.78 (d, J = 8.6 Hz, 2H, aromatic), 7.19 (d,
J = 8.6 Hz, 2H, aromatic);
13
C NMR (100 MHz, CDCl3): δ = -5.39, -5.34 (2C, Si(CH3)3), 11.5 (CH3(1)), 16.6 (CH3(2)),
18.2 (TBS quarternary), 25.9 (3C, tBu), 32.1 (CHCH2OH), 35.5 (CH2CH2OTBS), 37.5
(CHCH2CH2OTBS), 55.1 (OCH3), 61.5 (CH2CH2OTBS), 66.3 (CH2OH), 73.4 (CH2Ph), 84.2
(CHOPMB), 113.6, 129.1 (aromatic) 131.0 (Cipso aromatic), 159.0 (phenolic);
HRMS (EI): calcd for C22H40O4Si [M+Na]+: 419.25881, found 419.25891.
Experimental Section
195
tert-Butyl({(3R)-3-[(4S,5S)-2-(4-methoxyphenyl)-5-methyl-1,3-dioxan-4-yl]butyl}oxy)
dimethylsilane (224)
OMe
O
O
OTBS
2
1
To a solution of compound 231 (1.10 g, 3.92 mmol) in DMF (40 mL) was added imidazole
(670 mg, 9.80 mmol) at room temperature followed by the addition of TBDMS-Cl (770 mg,
5.10 mmol). After being stirred overnight, the mixture was diluted with water (40 mL). The
resultant mixture was stirred for 5 min. The mixture was extracted with diethylether (3 x 50
mL). The combined ether layers washed with 1 N HCl (40 mL), sat.NaHCO3 (40 mL) and then
with brine (40 mL). The organic layer dried over Na2SO4, filtered and concentrated in vacuo to
get the crude product. The crude product was purified by flash chromatography (5:95 ethyl
acetate/petroleum ether) to furnish the pure product 224 (1.30 g, 84%) as colorless oil.
Rf = 0.45 (5:95 ethyl acetate/petroleum ether);
[α]20D = +20.5 (c 1.00, CH2Cl2);
IR (film): νmax = 2950, 2850, 1620, 1515, 1250, 1100 cm-1;
1
H NMR (400 MHz, CDCl3): δ = 0.01 (s, 6H, Si(CH3)3), 0.86 (d, J = 6.6 Hz, 3H, CH3(1)),
0.87 (s, 9H, tBu), 1.13 (d, J = 7.1 Hz, 3H, CH3(2)), 1.32 (td, J = 14.0, 6.7 Hz,1H,
CH2CH2OTBS), 1.66 (qd, J = 6.8, 1.8 Hz, 1H, CHCH2O), 1.71-1.80 (m, 1H, CHCH2CH2),
1.97-2.05 (m, 1H, CH2CH2OTBS), 3.48 (dd, J = 10.0, 1.9 Hz, 1H, CHO), 3.68 (t, J = 7.2 Hz,
2H, CH2OTBS), 3.79 (s, 3H, OCH3), 3.99-4.05 (m, 2H, CH2O), 5.41 (s, 1H, CHO2), 6.87 (d, J
= 8.8 Hz, 2H, aromatic), 7.41 (d, J = 8.6 Hz, 2H, aromatic);
13
C NMR (100 MHz, CDCl3): δ = -5.28 (2C, Si(CH3)3), 10.6 (CH3(2)), 14.7 (CH3(1)), 18.3
(TBS quaternary), 26.0 (3C, tBu), 30.0 (CHCH2O), 31.6 (CHCH2CH2OH), 36.2
196
Experimental Section
(CH2CH2OTBS), 55.3 (OCH3), 61.8 (CH2CH2OTBS), 74.0 (CH2O), 84.2 (CHO), 101.4
(CHPh), 113.5, 127.3 (aromatic) 131.6 (Cipso aromatic), 159.7 (phenolic);
HRMS (EI): calcd for C22H38O4Si [M+H]+: 395.26121, found 395.24154.
(4S)-4-Benzyl-3-[(2S,3R,4R)-3-hydroxy-2,4-dimethylhept-6-enoyl]-1,3-oxazolidin-2-one
(226)
O
O
O
OH
N
Bn 2
1
A solution of oxalyl chloride (2.50 mL, 28.75 mmol) in dry CH2Cl2 (55 mL) was cooled to -78
o
C and a solution of DMSO (4.10 mL, 57.5 mmol) in dry CH2Cl2 (8 mL) was added dropwise
to the above cooled solution. After 5 min, a solution of alcohol 218 (2.60 g, 26.00 mmol) in
dry CH2Cl2 was added dropwise to the above solution at -78 oC. Stirring was continued for 30
min at -78 oC and Et3N (13.8 mL, 130 mmol) was added over 5 min, during which the solution
became a colorless heterogeneous mixture. At this point the cooling machine was switched off
and the reaction mixture slowly allowed to reach room temperature over 2.5 h. Water (50 mL)
was added to the reaction mixture and the layers were separated. The dichloromethane layer
was washed with 1 N HCl (4 x 20 mL), saturated NaHCO3 solution (20 mL), then with brine
(30 mL), and finally dried (Na2SO4). After filtration, the solution was partially concentrated by
keeping the water bath at 50 oC and pressure at 750 mbar until it became about 10 mL. This
solution was used for the aldol reaction without purification by taking the yield as 100%.
To a solution of chiral reagent ent-100 (3.60 g, 15.5 mmol) in CH2Cl2 (40 mL) was
added dropwise 1 M solution of n-butylboron triflate (18.6 mL, 18.6 mmol) at 0 oC, followed
by NEt3 (2.8 mL, 28.1 mmol). The resulting solution was cooled to -78 oC in 20-25 min. After
being stirred for 5 min at -78 oC, the crude solution of aldehyde was added to the reaction
mixture. The resulting reaction mixture was stirred for 3 h at -78 oC and then gradually
warmed to room temperature over a period of 2 h, and stirred at room temperature for 1.5 h.
The reaction mixture was treated with pH 7 aqueous phosphate buffer (30 mL) followed by a
2:1 mixture of MeOH and 30 wt% aqueous H2O2 (60 mL). The reaction mixture was stirred
Experimental Section
197
for 1 h. The reaction mixture was partially concentrated in vacuo and the resultant was
extracted with diethyl ether (3 x 50 mL). The combined ether layers were washed with
saturated NaHCO3 solution (40 mL), brine, dried (Na2SO4), filtered and concentrated in vacuo
to get the crude aldol product. The crude product was purified by flash chromatography (5:95
ethyl acetate/dichloromethane) to afford the pure aldol product 226 (4.0 g, 78%) as a colorless
gel.
Rf = 0.33 (5:95 ethyl acetate/dichloromethane);
[α]20D = +50.3 (c 1.10, CH2Cl2);
IR (film): νmax = 3510, 2970, 2850, 1780, 1690, 1385, 1210 cm-1;
1
H NMR (400 MHz, CDCl3): δ = 0.89 (d, J = 6.8 Hz, 3H, CH3(1)), 1.26 (d, J = 7.1 Hz, 3H,
CH3(2)), 1.65-1.75 (m, 1H, CHCH2CH2), 1.93-2.01 (m, 1H, CH2CH=CH2), 2.52-2.56 (m, 1H,
CH2CH=CH2), 2.81 (dd, J = 13.4, 9.4 Hz, 1H, PhCH2), 3.09 (d, J = 3.0 Hz, 1H, OH), 3.27 (dd,
J = 13.4, 3.0 Hz, 1H, PhCH2), 3.65 (d, J = 9.1 Hz, 1H, CHOH), 3.96 (qd, J = 7.0, 1.9 Hz,
COCH), 4.20 (dd, J = 9.2, 2.9 Hz, 1H, CH2O), 4.24 (m, 1H, CH2O), 4.69 (m, 1H, CHN), 5.04
(d, J = 9.9 Hz, CH2 olefinic cis), 5.07 (d, J = 16.9 Hz, CH2 olefinic trans), 5.78-5.88 (m, 1H,
CH olefinic), 7.22 (d, J = 7.1 Hz, 2H, aromatic), 7.30 (d, J = 7.1 Hz, 1H, aromatic), 7.35 (t, J =
7.2 Hz, 2H, aromatic);
13
C NMR (100 MHz, CDCl3): δ = 9.5 (CH3(2)), 15.1 (CH3(1)), 35.4 (CHCH2), 37.3
(CH2CH=CH2), 37.7 (PhCH2), 39.4 (COCH), 55.1 (CHN), 66.1 (CH2O), 74.6 (CHOH), 116.4
(CH2 olefin), 127.4, 128.9, 129.4, 135.0 (aromatic), 136.9 (CH olefin), 152.8 (NCO2), 177.9
(CO);
HRMS (EI): calcd for C19H25NO4 [M+Na]+ : 354.16758, found 354.16775.
(4S)-4-Benzyl-3-((2S,3R,4R)-3-{[tert-butyl(dimethyl)silyl]oxy}-6,7-dihydroxy-2,4dimethylheptanoyl)-1,3-oxazolidin-2-one
O
O
O
OTBS
OH
N
OH
Bn
198
Experimental Section
To a solution of protected aldol product 228 (4.50 g, 10.00 mmol), NMO (2.70 g, 20.00 mmol)
in a 8:1 mixture of THF and tert-BuOH (90 mL) was added a pre prepared catalytic solution of
osmium tetroxide in water (20 mL, 0.8 mmol) (prepared by dissolving K2OsO4.2H2O (240 mg,
0.8 mmol) in water (20 mL) to make the whole solution to 0.04 M). After being stirred for 12
h, the reaction mixture was treated with 10% aqueous Na2S2O3 solution (20 mL). The resultant
mixture was extracted with ethyl acetate (3 x 75 mL). The combined organic layers were dried
over Na2SO4, filtered and concentrated in vacuo to give the crude diol which was used for the
next step without further purification.
(3R,4R,5S)-6-[(4S)-4-Benzyl-2-oxo-1,3-oxazolidin-3-yl]-4-{[tert-butyl(dimethyl)silyl]oxy}3,5-dimethyl-6-oxohexanal
O
O
O
OTBS
H
N
O
Bn
To a solution of the above dihydroxylated product in a 9:1 mixture of THF and H2O (40 mL)
was added NaIO4 (6.40 g, 30.00 mmol) at room temperature. After being stirred for 1 h, the
reaction mixture was diluted with water (30 mL) and extracted with ether (3 x 40 mL). The
combined ether layers were washed with water (40 mL) and then with brine (40 mL). The
ether layer was dried over Na2SO4, filtered and concentrated in vacuo to furnish the crude
aldehyde which was used for the reduction step without further purification.
(2R,3R,4R)-3-{[tert-Butyl(dimethyl)silyl]oxy}-2,4-dimethylhexane-1,6-diol (229)
OTBS
OH
HO
1
2
Experimental Section
199
To a solution of the crude aldehyde in THF (200 mL) was added a solution of NaBH4 (2.62 g,
70.0 mmol) in H2O (50 mL) at 0 oC. After being stirred over night at room temperature, the
reaction was quenched by the addition of saturated NH4Cl solution (50 mL). The resulting
layers were separated and the aqueous layer extracted with ethyl acetate (3 x 75 mL). The
combined organic layers were washed with saturated NaHCO3 (50 mL) and brine (50 mL).
The organic layer was dried over Na2SO4, filtered and concentrated in vacuo to obtain the
crude diol which was purified using flash chromatography (1:4 ethyl acetate/dicloromethane)
leading to the pure diol 229 (2.20 g, 80% yield) as colorless oil.
Rf = 0.33 (1:4 ethyl acetate/dichloromethane);
[α]20D = -7.10 (c 1.06, CH2Cl2);
IR (film): νmax = 3340, 2925, 2850, 1770, 1460, 835 cm-1;
1
H NMR (400 MHz, CDCl3): δ = 0.07 (s, 6H, Si(CH3)2), 0.88 (d, J = 7.1 Hz, 3H, CH3(1)),
0.94 (d, J = 6.8 Hz, 3H, CH3(2)), 1.35-1.43 (m, 1H, CHCH2CH2), 1.73-1.92 (m, 3H,
CHCH2CH2, CHCH2OH), 2.06 (br s, 1H, OH), 3.47-3.66 (m, 4H, CH2OH, CH2CH2OH), 3.713.77 (m, 1H, CHOTBS);
13
C NMR (100 MHz, CDCl3): δ = -4.24, -3.95 (2C, Si(CH3)3), 12.6 (CH3(1)), 17.4 (CH3(2)),
18.4 (TBS quaternary), 26.0 (3C, tBu), 34.1 (CHCH2CH2), 35.5 (CH2CH2OH), 38.9
(CHCH2OH), 61.1 (CH2CH2OH), 66.2 (CH2OH), 78.1 (CHOTBS);
HRMS (EI): calcd for C14H32O3Si [M+Na]+: 299.20129, found 299.20110.
(2R,3R,4R)-2,4-Dimethylhexane-1,3,6-triol (230)
OH
HO
OH
To a solution of diol 229 (2.10 g, 7.60 mmol) in THF (35 mL) was added a 1 M solution of
TBAF in THF (19.0 mL, 19.0 mmol) at room temperature. The resulting reaction mixture was
stirred for 7 h at room temperature. The reaction mixture was evaporated to give the crude
200
product.
Experimental Section
The
crude
product
was
purified
by
flash
chromatography
(7:93
methanol/dichloromethane) to afford the pure triol 230 (1.01 g, 82%) as colorless oil.
Rf = 0.23 (7:93 methanol/dichloromethane);
1
H NMR (400 MHz, CDCl3): δ = 0.86 (d, J = 6.6 Hz, 3H, CH3(1)), 0.93 (d, J = 6.8 Hz, 3H,
CH3(2)), 1.40-1.45 (m, 1H, CH2CH2OTBS), 1.57-1.65 (m, 1H, CH2CH2OTBS), 1.70-1.80 (m,
2H, CHCH2CH2OH, CHCH2OH), 3.42-3.46 (m, 1H, CHOH), 3.54-3.76 (m, 4H, CH2OH,
CH2CH2OH), 4.41 (br s, 1H, OH);
13
C NMR (100 MHz, CDCl3): δ = 8.7 (CH3(2)), 17.5 (CH3(1)), 35.0 (CHCH2CH2), 35.9
(CHCH2OH), 37.6 (CH2CH2OH), 61.0 (CH2CH2OH), 67.8 (CH2OH), 78.3 (CHOH);
(3R)-3-[(4S,5S)-2-(4-Methoxyphenyl)-5-methyl-1,3-dioxan-4-yl]butan-1-ol (231)
OMe
O
O
OH
To a solution of triol 230 (1.00 g, 6.16 mmol) in CH2Cl2 (60 mL) was added pmethoxybenzaldehyde dimethyl acetal (1.26 mL, 6.78 mmol) at room temperature followed by
the addition of PPTS (154 mg, 0.62 mmol). The resulting mixture was stirred for 2 h at room
temperature. The reaction mixture was concentrated in vacuo and purified by flash
chromatography (1:3 ethyl acetate/petroleum ether) to get the pure product 231 (1.20 g, 72%)
as a colorless oil.
Rf = 0.45 (1:3 ethyl acetate/petroleum ether);
[α]20D = +21.6 (c 1.09, CH2Cl2);
IR (film): νmax = 3420, 2960, 2850, 1620, 1515, 1450, 1030 cm-1;
Experimental Section
1
201
H NMR (400 MHz, CDCl3): δ = 0.87 (d, J = 6.8 Hz, 3H, CH3(1)), 1.16 (d, J = 7.1 Hz, 3H,
CH3(2)), 1.46 (dt, J = 19.1, 6.1 Hz, 1H, CH2CH2OH), 1.64-1.70 (m, 1H, CHCH2CH2OH),
1.75-1.80 (m, 1H, CHCH2O), 2.09 (br s, 1H, OH), 3.51 (dd, J = 10.0, 2.2 Hz, 1H, CHO), 3.553.61 (m, 1H, CH2OH), 3.64-3.70 (m, 1H, CH2OH), 3.78 (s, 3H, OCH3), 4.02 (s, 2H, CH2O),
5.43 (s, 1H, CHPh), 6.87 (d, J = 8.8 Hz, 2H, aromatic), 7.40 (d, J = 8.8 Hz, 2H, aromatic);
13
C NMR (100 MHz, CDCl3): δ = 10.8 (CH3(1)), 15.5 (CH3(2)), 30.1 (CHCH2O), 32.3
(CHCH2CH2OH), 37.4 (CH2CH2OH), 55.2 (OCH3), 61.4 (CH2CH2OH), 73.9 (CH2OH), 84.2
(CHO), 101.9 (CHPh), 113.6, 127.3 (aromatic) 131.1 (Cipso aromatic), 159.9 (phenolic);
HRMS (EI): calcd for C16H24O4 [M+Na]+: 303.15668, found 303.15653.
(2R,3S,4R)-6-{[tert-Butyl(dimethyl)silyl]oxy}-3-[(4-methoxybenzyl)oxy]-2,4dimethylhexanal (232)
O
OPMB
H
OTBS
A solution of oxalyl chloride (0.23 mL, 2.52 mmol) in CH2Cl2 (5 mL) was cooled to -78 oC.
To this solution, a solution of DMSO (0.37 mL, 5.04 mmol) in CH2Cl2 (0.5 mL) was added
dropwise. After stirring for 15 min, a solution of alcohol 222 (900 mg, 2.27 mmol) was added
drop wise to the above mixture at -78 oC. After the reaction mixture was stirred for 1 h,
triethylamine (1.3 mL, 9.43 mmol) was added dropwise at -78 oC and the mixture allowed to
reach room temperature over the course of 4 h. Then the mixture was diluted with water (4
mL) and CH2Cl2 (4 mL). The organic layer was washed with 1 N HCl (5 mL), saturated
aqueous NaHCO3 solution (5 mL). The organic layer was dried over MgSO4, filtered, and
concentrated in vacuo. The crude product was filtered through a short pad of silica gel (1:3
ethyl acetate/petroleum ether) to afford aldehyde (800 mg, 89%) as colorless oil.
Rf = 0.40 (1:3 ethyl acetate/petroleum ether);
1
H NMR (400 MHz, CDCl3): δ = 0.03 (s, 6H, Si(CH3)3), 0.86 (s, 9H, tBu), 0.88 (d, J = 6.8
Hz, 3H, CH3), 1.16 (d, J = 6.8 Hz, CH3CHCHO), 1.78-1.97 (m, 3H, CHCH2CH2,
202
Experimental Section
CH2CH2OTBS), 2.56-2.62 (m, 1H, CHCHO), 3.59-3.72 (m, 3H, CHOPMB, CH2OTBS), 3.79
(s, 3H, OCH3), 4.37 (d, J = 10.9 Hz, 1H, CH2Ph), 4.41 (d, J = 10.9 Hz, 1H, CH2Ph), 6.85 (d, J
= 8.6 Hz, 2H, aromatic), 7.20 (d, J = 8.6 Hz, 2H, aromatic), 9.76 (s, 1H, CHO);
13
C NMR (100 MHz, CDCl3): δ = -5.35, -5.30 (2C, Si(CH3)3), 8.4 (CH3CHCHO), 16.3 (CH3),
18.3 (TBS quarternary), 25.9 (3C, tBu), 32.8 (CHCH2CH2), 35.5 (CH2CH2OTBS), 49.0
(CHCHO), 55.3 (OCH3), 61.3 (CH2CH2OTBS), 73.1 (CH2Ph), 84.2 (CHOPMB), 113.7, 129.2
(aromatic) 131.4 (Cipso aromatic), 159.2 (phenolic), 204.8 (CHO).
7-O-[tert-Butyl(dimethyl)silyl]-3,5,6-trideoxy-4-O-(4-methoxybenzyl)-3,5-dimethyl-Dgluco-heptitol (diastereomer-233)
OH OPMB
HO
OTBS
To a solution of olefin 212 (25 mg, 0.064 mmol) in a mixture of 1:1 H2O and tert-butanol (1.0
mL) was added AD-mix-α (90 mg) at 0 oC. Stirring was continued for 3 d at 0 oC. The reaction
was quenched with 10% Na2S2O3 solution (1 mL) at 0 oC. The mixture was extracted with
ethyl acetate (3 x 5 mL). The combined organic layers were dried (Na2SO4), filtered and
concentrated to give the crude diol in 4:1 diastereomeric mixture which was purified by flash
chromatography (1:1 ethylacetate/petroleum ether) to get the pure diol diastreomer-233 (5
mg, yield was not determined because of very small amounts) as an oil.
Rf = 0.50 (1:1 ethyl acetate/petroleum ether);
1
H NMR (400 MHz, CDCl3): δ = 0.03 (s, 6H, Si(CH3)3), 0.88 (s, 12H, tBu, CH3CHCH2), 0.91
(d, J = 7.1 Hz, 3H, CH3CHCH), 1.30-1.44 (m, 2H, CH2CH2OTBS), 1.90-1.77 (m, 2H,
CHCHOH, CHCH2CH2), 2.56-2.62 (m, 1H, CHCH2O), 3.47-3.51 (m, 2H, CHOPMB, CHOH),
3.60-3.73 (m, 4H, CH2OH, CH2OTBS), 3.79 (s, 3H, OCH3), 4.55 (s, 2H, CH2Ph), 6.86 (d, J =
8.6 Hz, 2H, aromatic), 7.26 (d, J = 7.3 Hz, 2H, aromatic);
13
C NMR (100 MHz, CDCl3): δ = -5.34, -5.28 (2C, Si(CH3)3), 11.4 (CH3CHCH), 16.3
(CH3CHCH2), 18.3 (TBS quarternary), 26.0 (3C, tBu), 31.6 (CHCH2CH2), 35.8
Experimental Section
203
(CH2CH2OTBS), 36.9 (CHCHOH), 55.3 (OCH3), 61.5 (CH2CH2OTBS), 73.1 (CH2Ph), 84.1
(CHOPMB), 113.8, 129.2 (aromatic) 131.4 (Cipso aromatic), 159.1 (phenolic).
204
9 Appendix
9.1 NMR-Spectra for important compounds
Appendix
Appendix
205
O
BocHN
OH
95
7.5
7.0
6.5
6.0
5.5
5.0
4.5
4.0
ppm
3.5
3.0
2.5
2.0
1.5
1.0
0.5
150
100
ppm
50
20.52
17.17
28.79
43.83
42.69
40.67
79.77
140.75
140.33
131.20
129.25
128.40
127.92
157.75
179.98
Methanol-d4
206
Appendix
HO2C
OR
96 R = TBDPS
8.0
7.5
7.0
6.5
6.0
5.5
5.0
4.5
4.0
ppm
3.5
3.0
2.5
2.0
1.5
1.0
150
100
ppm
50
26.82
19.23
17.31
16.19
43.77
37.75
35.41
68.51
77.31
77.00
76.68
135.61
133.97
132.03
130.78
129.49
127.55
182.81
Chloroform-d
Appendix
207
HO2C
NHR
97
4.5
4.0
ppm
150
3.5
100
ppm
3.0
2.5
2.0
50
1.5
1.0
18.89
17.00
16.87
5.0
38.55
33.70
29.08
5.5
47.14
44.30
6.0
79.75
77.69
6.5
134.14
131.52
7.0
156.70
182.40
7.5
208
Appendix
O
O
HO
OH
98
12
11
10
9
8
7
6
5
4
3
2
1
ppm
150
100
ppm
50
16.32
41.27
39.13
77.32
77.00
76.69
129.76
128.45
127.07
139.04
182.79
Chloroform-d
Appendix
209
O
O
MeO
OH
103
7.5
7.0
6.5
6.0
5.5
5.0
4.5
4.0
ppm
3.5
3.0
2.5
2.0
1.5
1.0
0.5
150
100
ppm
50
16.63
41.39
39.57
39.18
51.55
77.32
77.00
76.68
129.67
128.39
126.95
139.38
139.04
176.58
182.25
Chloroform-d
210
Appendix
N
N
Bn
6.0
5.5
5.0
4.5
4.0
ppm
3.5
3.0
2.5
2.0
Chloroform-d
150
100
ppm
1.5
50
1.0
0.5
16.59
108
139.18
135.22
130.40
129.34
128.84
128.28
127.33
127.20
6.5
152.96
176.46
7.0
O
39.67
39.47
37.68
Bn
7.5
O
O
65.84
O
O
55.09
O
150
5.5
5.0
4.5
4.0
ppm
100
ppm
3.5
3.0
2.5
2.0
50
1.5
20.10
16.66
6.0
28.38
6.5
51.56
47.44
42.85
41.37
39.58
BocHN
79.09
77.32
77.00
76.69
7.0
139.33
138.26
130.18
129.83
128.30
127.49
126.95
120.13
7.5
155.15
176.52
Appendix
211
O
OMe
115
1.0
Chloroform-d
150
6
5
100
ppm
4
3
2
50
20.63
17.24
7
49.64
49.43
49.21
49.00
48.79
48.57
48.36
43.25
42.33
40.07
28.79
HO
79.77
8
131.28
130.73
130.08
122.93
9
143.01
142.62
157.52
179.40
212
Appendix
O
NHBoc
Br
116
ppm
1
Methanol-d4
150
Bn
6.0
5.5
5.0
4.5
4.0
ppm
100
ppm
3.5
3.0
2.5
50
2.0
16.64
N
39.45
39.23
37.73
N
55.11
6.5
O
65.93
O
O
77.31
77.00
76.68
O
141.46
135.15
130.24
129.32
128.87
127.25
122.22
7.0
152.96
176.04
Appendix
213
O
O
Br
119
Bn
1.5
1.0
Chloroform-d
214
Appendix
Ph
H
CO2Me
N
O
H
N
Boc
126
7.5
7.0
6.5
6.0
5.5
5.0
4.5
4.0
ppm
3.5
3.0
2.5
2.0
1.5
1.0
150
100
ppm
50
18.38
28.25
37.85
53.01
52.28
49.88
80.01
135.70
129.20
128.53
127.08
155.33
172.22
171.74
Chloroform-d
Appendix
215
Ph
H
CO2Me
N
Me
H
O
O
N
N
H
Boc
127
7.5
7.0
6.5
6.0
5.5
5.0
4.5
4.0
ppm
3.5
3.0
2.5
2.0
1.5
1.0
0.5
150
100
ppm
50
18.35
18.09
28.25
37.82
53.17
52.28
50.17
48.50
80.07
129.22
128.48
127.03
135.85
155.36
172.57
171.81
Chloroform-d
216
Appendix
DMSO-d6
Ph
H
Me
O
N
N
N
O
O
Me
N
H
H
H
O
129
7.0
6.5
6.0
5.5
5.0
4.5
4.0
ppm
3.5
3.0
2.5
2.0
1.5
HMQC spectrum of macrocycle 129 (320 K, 600 MHz, DMSO-d6).
1.0
Appendix
217
Ph
H
CO2Me
N
O
Me
N
Boc
131
7.5
7.0
6.5
6.0
5.5
5.0
4.5
4.0
ppm
3.5
3.0
2.5
2.0
1.5
1.0
150
100
50
ppm
13.24
29.65
28.25
37.77
52.90
52.20
80.54
135.69
129.08
128.61
127.11
171.71
171.31
Chloroform-d
N
8.0
7.5
7.0
150
6.5
6.0
5.5
5.0
4.5
ppm
100
ppm
4.0
55.56
53.81
52.70
47.28
43.84
43.04
40.74
38.07
31.42
28.79
20.87
20.60
17.43
16.73
13.96
8.5
N
CO2Me
79.72
Me
N
130.25
129.45
129.23
128.29
127.98
127.81
H
140.82
140.36
138.43
Me
157.69
178.70
175.27
173.43
173.16
218
Appendix
Ph
NHBoc
O
O
H
O
133
3.5
3.0
50
2.5
2.0
1.5
1.0
Methanol-d4
0
0.5
Appendix
219
H2O
Ph
H
O
N
N
Me
N
O
O
Me
N
Me
DMSO-d6
H
H
O
134
8.5
8.0
7.5
7.0
6.5
6.0
5.5
5.0
4.5
ppm
4.0
3.5
3.0
2.5
2.0
1.5
HMQC spectrum of macrocycle 134 (300 K, 600 MHz, DMSO-d6).
1.0
150
5.5
5.0
4.5
4.0
ppm
100
ppm
3.5
3.0
2.5
50
2.0
1.5
11.22
9.20
6.0
27.73
26.81
19.20
19.02
6.5
37.33
7.0
77.32
77.00
76.69
76.50
65.37
7.5
112.01
8.0
142.30
135.58
133.70
133.58
129.59
127.60
173.38
220
Appendix
O
O
OR
R = TBDPS 140
1.0
Chloroform-d
Appendix
221
OH
OH
143
5.5
5.0
4.5
4.0
3.5
3.0
ppm
2.5
2.0
1.5
1.0
0.5
130
120
110
90
80
ppm
70
37.19
66.82
77.44
77.12
77.00
76.80
100
60
50
40
30
9.81
140
19.42
160 150
110.62
146.33
Chloroform-d
20
10
222
Appendix
OH
OTBDPS
144
8.0
7.5
7.0
6.5
6.0
5.5
5.0
4.5
4.0
ppm
3.5
3.0
2.5
2.0
1.5
1.0
0.5
150
140
130
120
110
100
90
80
ppm
70
60
50
40
30
20
9.71
19.38
19.19
26.85
37.34
67.95
76.29
110.53
135.67
134.78
133.12
129.73
127.72
145.73
Chloroform-d
10
Appendix
223
OR O
NH2
154 R = TBS
7
6
5
4
3
2
1
0
ppm
150
100
50
ppm
-4.82
12.45
18.01
25.77
45.36
77.81
77.32
77.00
76.69
113.21
144.69
177.22
Chloroform-d
0
224
Appendix
OR O
OH
155 R = TBS
11
10
9
8
7
6
5
4
3
2
1
0
ppm
150
100
50
ppm
-4.65
11.34
25.74
18.14
17.74
44.33
77.46
77.32
77.00
76.68
113.16
144.68
180.79
Chloroform-d
0
Appendix
225
O
O
NHR
147 R = Boc
5.5
5.0
4.5
4.0
3.5
3.0
ppm
2.5
2.0
1.5
1.0
150
100
50
ppm
11.82
9.19
19.30
28.36
27.71
35.14
43.21
79.18
77.31
77.00
76.68
76.41
112.10
141.92
155.95
173.96
Chloroform-d
226
Appendix
OH
NHR
158 R = Boc
5.5
5.0
4.5
4.0
3.5
3.0
2.5
2.0
1.5
1.0
0.5
0.0
ppm
130
120
110
90
80
ppm
70
60
50
40
28.35
36.31
43.80
79.57
77.32
77.00
76.69
74.08
100
30
10.60
140
19.54
150
110.49
145.67
157.07
Chloroform-d
20
10
6.5
150
6.0
5.5
Boc
5.0
4.5
4.0
3.5
ppm
100
ppm
3.0
2.5
2.0
50
1.5
1.0
0.5
0
-4.50
N
H
43.90
38.58
32.84
31.97
30.57
28.27
28.07
25.64
19.09
18.17
17.55
16.77
15.95
7.0
N
O
O
57.38
57.19
HN
79.87
79.55
77.31
77.00
76.68
69.50
TBSO
134.38
129.74
129.35
128.15
120.02
7.5
155.31
154.35
175.72
174.52
171.69
170.12
Appendix
227
O
O
O
OtBu
161
0.0
Chloroform-d
150
3.0
100
ppm
2.5
2.0
50
1.5
19.29
17.49
17.11
16.76
3.5
32.05
28.06
4.0
38.63
4.5
43.87
5.0
59.89
5.5
79.88
77.32
77.00
76.69
69.07
H2N
128.42
6.0
134.23
175.74
175.50
228
Appendix
O
O
O
tBuO
163
ppm
1.0
Chloroform-d
150
6.5
6.0
5.5
5.0
4.5
4.0
ppm
100
ppm
3.5
3.0
2.5
2.0
50
38.65
35.37
28.06
26.82
19.23
17.42
16.86
15.92
7.0
44.07
7.5
68.55
ButO2C
79.72
77.31
77.00
76.68
8.0
135.61
133.99
132.84
129.81
129.46
127.54
175.91
Appendix
229
OR
169 R = TBDPS
1.5
1.0
0.5
Chloroform-d
21
20
19
150
18
ppm
4.5
17
4.0
3.5
ppm
100
ppm
3.0
2.5
2.0
50
1.5
1.0
0.5
-4.49
5.0
32.78
30.82
30.43
28.41
25.64
19.04
18.23
17.88
17.44
16.90
O
O
57.51
57.08
52.06
46.50
45.53
43.67
5.5
77.91
77.32
77.00
76.68
6.0
16.39
HN
16.90
6.5
N
17.44
7.0
130.50
130.45
129.71
129.31
120.05
TBSO
18.23
18.17
17.88
19.04
7.5
156.05
154.36
175.74
174.33
172.25
170.05
230
Appendix
CO2Me
NHBoc
NH
O
181
0.0
Chloroform-d
16
0
150
6
5
4
ppm
100
ppm
3
2
50
1
-4.55
N
H
46.55
32.70
30.75
30.38
28.20
25.59
18.99
18.12
17.76
17.45
N
57.48
57.12
52.01
HN
79.63
77.32
77.00
76.69
TBSO
119.99
7
129.69
129.25
8
155.38
154.32
174.82
172.04
170.16
Appendix
231
CO2Me
O
O
Boc
182
0
Chloroform-d
0
N
8
7
150
N
6
5
4
100
ppm
3
2
50
-4.47
Me
N
60.36
56.54
51.69
49.41
46.59
40.48
30.67
28.28
25.61
23.10
21.00
18.11
17.06
14.14
H
79.74
77.32
77.00
76.68
H
136.07
133.26
127.33
122.03
121.92
120.05
119.30
118.50
111.04
110.94
155.52
154.92
174.33
171.14
169.24
232
Appendix
OTBS
O
OMe
O
O
H
N
Boc
184
ppm
1
0
Chloroform-d
0
19.5
19.0
150
18.5
N
N
6
5
18.0
ppm
17.5
4
17.0
100
ppm
3
2
16.5
50
23.87
18.91
18.02
17.55
Me
O
O
49.74
45.69
42.51
41.13
40.38
31.83
N
56.25
H
69.57
7
17.55
8
136.15
135.38
132.23
127.20
127.02
125.67
122.45
122.07
119.50
118.44
115.59
111.23
110.15
H
18.08
18.02
18.91
155.64
175.29
174.28
170.86
169.45
Appendix
233
OH
O
N
O
H
O
185
ppm
1
Chloroform-d
N
Me
8
7
150
N
N
H
N
Boc
6
O
5
4
ppm
100
ppm
3
2
50
1
-4.48
H
79.82
77.32
77.00
76.68
68.80
60.35
56.63
49.43
46.58
43.85
38.57
31.90
30.76
28.29
28.03
26.51
25.61
18.11
17.38
16.95
16.71
15.90
136.05
134.77
134.16
133.31
129.49
128.24
127.61
127.42
121.91
120.02
119.30
118.50
111.03
H
155.55
154.90
175.72
174.27
170.73
169.17
234
Appendix
OTBS
O
O
O
O
193
OtBu
0
Chloroform-d
0
Appendix
235
O
HO
NHR
194 R = Boc
8.0
7.5
7.0
6.5
6.0
5.5
5.0
4.5
ppm
4.0
3.5
3.0
2.5
2.0
1.5
150
100
ppm
50
16.51
28.38
44.59
41.10
39.22
79.57
139.52
138.96
128.66
127.99
127.83
125.56
155.31
181.26
Chloroform-d
150
5
100
ppm
4
3
50
2
-5.25
6
25.93
18.40
16.41
7
41.15
39.21
8
64.92
9
77.32
77.00
76.69
HO
128.29
127.56
126.72
124.20
10
141.52
138.93
182.52
236
Appendix
O
OTBS
195
ppm
1
0
Chloroform-d
0
6.0
150
5.5
5.0
4.5
4.0
ppm
100
ppm
3.5
3.0
2.5
50
2.0
1.5
16.75
6.5
39.64
39.36
37.66
33.53
7.0
55.06
7.5
N
65.88
O
77.31
77.00
76.68
O
153.04
139.83
137.80
135.11
129.92
129.47
129.33
128.85
127.23
127.15
176.28
Appendix
237
O
Br
Bn
203
1.0
Chloroform-d
150
5.0
4.5
4.0
100
ppm
3.5
3.0
2.5
2.0
50
1.5
16.63
5.5
28.37
6.0
44.62
39.69
39.51
37.69
6.5
55.11
7.0
N
65.89
O
79.42
O
139.56
138.91
135.09
129.36
128.90
128.64
128.31
127.30
125.61
7.5
155.83
153.03
176.46
238
Appendix
O
NHBoc
Bn
203
ppm
1.0
Chloroform-d
150
100
ppm
3.0
2.5
50
2.0
1.5
1.0
-5.28
3.5
ppm
25.96
18.31
17.23
15.92
4.0
40.87
34.53
32.52
4.5
55.25
5.0
61.64
5.5
77.32
77.00
76.69
74.45
6.0
87.96
6.5
113.82
113.64
7.0
131.29
129.10
7.5
142.45
158.97
Appendix
239
OPMB
OTBS
213
0.5
0.0
Chloroform-d
0
150
100
ppm
3.0
2.5
50
2.0
1.5
1.0
0.5
-5.34
3.5
ppm
11.47
4.0
18.24
16.62
4.5
25.90
5.0
37.49
35.49
32.12
5.5
55.14
6.0
61.51
6.5
84.30
77.32
77.00
76.68
73.68
66.30
7.0
113.64
130.99
129.09
158.97
240
Appendix
OH OPMB
OTBS
223
0.0
Chloroform-d
0
150
5.0
4.5
4.0
ppm
100
ppm
3.5
3.0
2.5
50
2.0
1.5
9.54
5.5
15.10
6.0
39.35
37.69
37.27
35.36
6.5
55.06
7.0
66.10
Bn
77.32
77.00
76.68
74.64
O
O
116.36
O
136.93
134.96
129.36
128.90
127.37
7.5
152.80
177.86
Appendix
241
OH
N
226
1.0
Chloroform-d
242
Appendix
OH OTBS
OH
229
4.0
3.5
3.0
2.5
2.0
1.5
1.0
0.5
0.0
-0.5
ppm
140
130
120
110
100
90
80
70
ppm
60
50
40
30
20
10
-3.95
-4.24
12.62
18.31
17.36
26.02
38.89
35.51
34.14
61.14
66.15
77.31
77.00
76.68
Chloroform-d
0
-10
150
100
ppm
3.5
3.0
2.5
50
2.0
1.5
10.80
4.0
ppm
15.46
4.5
37.41
32.30
30.08
5.0
55.22
5.5
61.35
6.0
77.32
77.00
76.69
73.90
6.5
84.62
7.0
101.86
O
113.61
7.5
131.07
127.30
159.91
Appendix
243
PMP
O
OH
231
1.0
Chloroform-d
244
Appendix
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My academic teachers were:
K. Ananthaiah, K. Anjaneyulu, Sk. Badruddin, D. Basavaiah, S. K. Das, E. D. Jemmis, S.
Mahapatra, Micheal, M. E. Maier, M. Periasamy, A. J. Pratap Reddy, N. S. Prasad T. P.
Radhakrishnan, K. Rama Rao, K. Sagar, Samudranil Pal, B. Venkateswara Rao, Th. Ziegler.
CURRICULUM VITAE
Name:
Srinivasa Marimganti
Date of Birth:
August 22, 1980
Place of Birth:
Jaggiahpet, India
1989-1996
Schooling, Mahatma Gandhi Harijan High School, Nandigama, Andhra
Pradesh, India.
1996-1998
Higher Secondery Education, Jaggaiahpet Junior College, Jaggiahpet,
Andhra Pradesh, India.
1998-2001
B.Sc., in Mathematics, Physics, Chemistry, K. V. R. College,
Nandigama, Andhra Pradesh, India.
2001-2003
M.Sc., Chemistry, University of Hyderabad, India.
Master thesis with the title ‘Synthesis of Chiral Amino Naphthols’ under
the supervision of Prof. M. Periasamy, University of Hyderabad, India.
2003-2006
Ph.D., Organic chemistry, Universität Tübingen, Tübingen, Germany.
Doctoral thesis with the title ‘Synthesis and Conformational Analysis of
Jasplakinolide Analogues and Approach towards the Synthesis of the
Stereotetrad of Cruentaren A’ under the supervision of Prof. Dr. Martin
E. Maier, Eberhard-Karls-Universität, Tübingen, Germany.